Risk markers for cardiovascular disease

ABSTRACT

Provided herein methods for determining whether a subject, particularly a human subject, is at risk of developing, having, or experiencing a complication of cardiovascular disease, and methods of treating subjects who are identified by the current methods of being at risk for cardiovascular disease. In one embodiment, the method comprises determining levels of one or more oxidized apolipoprotein A-I related biomolecules in a bodily sample from the subject. Also, provided are kits and reagents for use in the present methods. Also provided are methods for monitoring the status of cardiovascular disease in a subject or the effects of therapeutic agents on subjects with cardiovascular disease. Such method comprising determining levels of one or more oxidized apolipoprotein A-I related molecules in bodily samples taken from the subject over time or before and after therapy.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.11/005,563, filed Dec. 6, 2004, which claims the benefit of U.S.Provisional Application No. 60/527,178, filed Dec. 5, 2003, U.S.Provisional Application No. 60/600,527, filed Aug. 11, 2004, U.S.Provisional Application No. 60/600,551, filed Aug. 11, 2004, and U.S.Provisional Application No. 60/619,044, filed Oct. 15, 2004, all ofwhich are incorporated herein by reference in their entirety.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH

The work described in this application was supported, at least in part,by Grant Nos. HL62526, HL076491, HL70621, HL077692, and HL66082. TheUnited States government has certain rights in this invention.

FIELD

The present invention relates to the field of cardiovascular disease.More specifically, it relates to markers and methods for determiningwhether a subject, particularly a human subject, is at risk ofdeveloping cardiovascular disease, having cardiovascular disease, orexperiencing a complication of cardiovascular disease. The presentapplication also relates to the use of such markers and methods formonitoring the status of cardiovascular disease in a subject or theeffects of therapeutic agents on subjects with cardiovascular disease.

Cardiovascular disease (CVD) is the general term for heart and bloodvessel diseases, including atherosclerosis, coronary heart disease,cerebrovascular disease, aorto-iliac disease, and peripheral vasculardisease. Subjects with CVD may develop a number of complications,including, but not limited to, myocardial infarction, stroke, anginapectoris, transient ischemic attacks, congestive heart failure, aorticaneurysm and death. CVD accounts for one in every two deaths in theUnited States and is the number one killer disease. Thus, prevention ofcardiovascular disease is an area of major public health importance.

A low-fat diet and exercise are recommended to prevent CVD. In addition,a number of therapeutic agents may be prescribed by medicalprofessionals to those individuals who are known to be at risk fordeveloping or having CVD. These include lipid-lowering agents thatreduce blood levels of cholesterol and trigylcerides, agents thatnormalize blood pressure, agents, such as aspirin or platelet ADPreceptor antatoginist (e.g., clopidogrel and ticlopidine) that preventactivation of platelets and decrease vascular inflammation, andpleotrophic agents such as peroxisome proliferator activated receptor(PPAR) agonists, with broad-ranging metabolic effects that reduceinflammation, promote insulin sensitization, improve vascular function,and correct lipid abnormalities. More aggressive therapy, such asadministration of multiple medications or surgical intervention may beused in those individuals who are at high risk. Since CVD therapies mayhave adverse side effects, it is desirable to have methods foridentifying those individuals who are at risk, particularly thoseindividuals who are at high risk, of developing or having CVD.

Currently, several risk factors are used by medical professionals toassess an individual's risk of developing or having CVD and to identifyindividuals at high risk. Major risk factors for cardiovascular diseaseinclude age, hypertension, family history of premature CVD, smoking,high total cholesterol, low HDL cholesterol, obesity and diabetes. Themajor risk factors for CVD are additive, and are typically used togetherby physicians in a risk prediction algorithm to target those individualswho are most likely to benefit from treatment for CVD. These algorithmsachieve a high sensitivity and specificity for predicting risk of CVDwithin 10 years. However, the ability of the present algorithms topredict a higher probability of developing CVD is limited. Among thoseindividuals with none of the current risk factors, the 10-year risk fordeveloping CVD is still about 2%. In addition, a large number of CVDcomplications occur in individuals with apparently low to moderate riskprofiles, as determined using currently known risk factors. Thus, thereis a need to expand the present cardiovascular risk algorithm toidentify a larger spectrum of individuals at risk for or affected withCVD.

The mechanism of atherosclerosis is not well understood. Over the pastdecade a wealth of clinical, pathological, biochemical and genetic datasupport the notion that atherosclerosis is a chronic inflammatorydisorder. Acute phase reactants (e.g. C-reactive protein, complementproteins), sensitive but non-specific markers of inflammation, areenriched in fatty streaks and later stages of atherosclerotic lesions.In a recent prospective clinical trial, base-line plasma levels ofC-reactive protein independently predicted risk of first-time myocardialinfarction and stroke in apparently healthy individuals. U.S. Pat. No.6,040,147 describes methods which use C-reactive protein, cytokines, andcellular adhesion molecules to characterize an individual's risk ofdeveloping a cardiovascular disorder. Although useful, these markers maybe found in the blood of individuals with inflammation due to causesother than CVD, and thus, these markers may not be specific enough.Moreover, modulation of their levels has not been shown to predict adecrease in the morbidity or mortality of CVD.

The present invention provides methods for characterizing a subject's,particularly a human subject's, risk of having cardiovascular disease.The present invention also provides methods of characterizing asubject's risk of developing cardiovascular disease. In anotherembodiment, the present invention provides methods for characterizing asubject's risk of experiencing a complication of cardiovascular disease.In another embodiment, the present invention provides a method fordetermining whether a subject presenting with chest pain is at risk nearterm of experiencing a heart attack or other major adverse cardiacevent. The present methods are especially useful for identifying thosesubjects who are in need of highly aggressive CVD therapies as well asthose subjects who require no therapies targeted at inhibiting orpreventing CVD or complications of CVD.

In one embodiment, the present methods comprise determining the levelsof one or more oxidized biomolecules (referred to hereinaftercollectively as oxidized “apolipoprotein Al-related biomolecules”) in abodily sample obtained from the subject. In one embodiment the oxidizedapolipoprotein Al (“apoA-I”)-related biomolecule is oxidizedhigh-density lipoprotein “HDL”. In another embodiment, the oxidizedapoA-I-related biomolecule is oxidized apoA-I. In another embodiment,the oxidized apoA-I-related biomolecule is an oxidized apoA-I peptidefragment. Levels of one or more of the oxidized apoA-I-relatedbiomolecules in a biological sample from the subject may be compared toa control value that is derived from measurements of the one or moreoxidized apoA-I-related biomolecules in comparable biological samplesobtained from a population of control subjects. Levels of the one ormore oxidized apoA-I-related biomolecules in a biological sampleobtained from the subject, alternatively, may be compared to levels ofan oxidized internal standard in the biological sample obtained from thesubject. Examples of such internal standards include, but are notlimited to, oxidized albumin or oxidized total protein.

In one embodiment, the comparison characterizes the subject's presentrisk of having CVD, as determined using standard protocols fordiagnosing CVD. Moreover, the extent of the difference between thesubject's oxidized apoA-I-related biomolecule levels and the controlvalue is also useful for characterizing the extent of the risk andthereby, determining which subjects would most greatly benefit fromcertain therapies. In another embodiment, the comparison characterizesthe subject's risk of developing CVD in the future. In anotherembodiment, the comparison can be used to characterize the subject'srisk of experiencing a complication of CVD. The present methods can alsobe used to determine if a subject presenting with chest pain is at riskof experiencing a major adverse cardiac event, such as a myocardialinfarction, reinfarction, the need for revascularization, or death, nearterm, e.g., within the following day, 3 months or 6 months after thesubject presents with chest pain.

Also provided herein are methods for monitoring over time the status ofCVD in a subject. In one embodiment, the method comprises determiningthe levels of one or more of the oxidized apoA-I-related biomolecules ina biological sample taken from the subject at an initial time and in acorresponding biological sample taken from the subject at a subsequenttime. An increase in levels of the one or more oxidized apoA-I-relatedbiomolecules in a biological sample taken at the subsequent time ascompared to the initial time indicates that a subject's risk of havingCVD has increased. A decrease in levels of the one or more oxidizedapoA-I-related molecules indicates that the subject's risk of having CVDhas decreased. For those subjects who have already experienced an acuteadverse cardiovascular event such as a myocardial infarction or ischemicstroke, such methods are also useful for assessing the subject's risk ofexperiencing a subsequent acute adverse cardiovascular event. In suchsubjects, an increase in levels of the one more oxidized apoA-I-relatedbiomolecules indicates that the subject is at increased risk ofexperiencing a subsequent adverse cardiovascular event. A decrease inlevels of the one or more oxidized apoA-I-related biomolecules in thesubject over time indicates that the subject's risk of experiencing asubsequent adverse cardiovascular event has decreased.

In another embodiment, the present invention provides a method forcharacterizing a subject's response to therapy directed at stabilizingor regressing CVD. The method comprises determining levels of one ormore oxidized apoA-I-related biomolecules in a biological sample takenfrom the subject prior to therapy and determining the level of the oneor more of the oxidized apoA-I related biomolecules in a correspondingbiological sample taken from the subject during or following therapy. Adecrease in levels of the one or more oxidized apoA-I-relatedbiomolecules in the sample taken after or during therapy as compared tolevels of the one or more oxidized apoA-I-related biomolecules in thesample taken before therapy is indicative of a positive effect of thetherapy on cardiovascular disease in the treated subject.

In another embodiment, the present invention provides antibodies thatare immunospecific for one or more of the oxidized apoA-I relatedbiomolecules used in the present methods. Such antibodies are useful fordetermining or measuring the levels of the apoA-I-related biomoleculesin biological samples obtained from the subject.

In another embodiment, the present invention relates to kits thatcomprise reagents for assessing levels of oxidized HDL, oxidized apoA-I,and/or oxidized apoA-I peptide fragments in biological samples obtainedfrom a test subject. The present kits also comprise printed materialssuch as instructions for practicing the present methods, or informationuseful for assessing a test subject's risk of CVD. Examples of suchinformation include, but are not limited cut-off values, sensitivitiesat particular cut-off values, as well as other printed material forcharacterizing risk based upon the outcome of the assay. In someembodiments, such kits may also comprise control reagents, e.g. oxidizedHDL, oxidized apoA-I, and/or oxidized apoA-I peptide fragments.

In another embodiment, the present invention relates to methods oftreating a subject to reduce the risk of cardiovascular disease.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1. Collision-induced dissociation (CID) spectra of thenitrotyrosine-containing peptides. The spectra were acquired during theanalysis of in-gel tryptic digests of the apoA-I band from HDL treatedwith either the MPO/H₂O₂/NO₂ ⁻ protein nitration system (A-D) orperoxynitrite (E), as described in the methods. Doubly and triplycharged ions (as indicated) were detected and fragmented in an LC-tandemMS experiment using an ion trap mass spectrometer system. The peptidessequenced in spectra A and D were detected in the MPO-mediated reactiononly. The peptides sequenced in spectra B and C were detected in boththe MPO-mediated and peroxynitrite-mediated reactions. The peptidesequenced in spectrum E was detected only in the peroxynitrite-mediatedreaction (SEQ ID NOs: 4, 5, 7, 2, and 8, respectively, in order ofappearance.).

FIG. 2. Collision-induced dissociation (CID) spectra of thechlorotyrosine-containing peptides. The spectra were acquired during theanalysis of in-gel tryptic digests of the apoA-I band from HDL treatedwith either the MPO/H₂O₂/Cl⁻ protein chlorination system, as describedin the methods. Doubly and triply charged ions (as indicated) weredetected and fragmented in an LC-tandem MS experiment using an ion trapmass spectrometer system. The same series of peptides were sequenced inboth the MPO- and HOCl-mediated reactions (SEQ ID NOs: 4, 5, 7, and 2,respectively, in order of appearance.).

FIG. 3. Summary of the apoA-I modification sites. The specific tyrosineresidues that were modified by the respective reactions are indicated.The amino acid sequence (SEQ ID NO: 1) is based on NCBI accession number229513 for the mature apoA-I protein. The numbering of all amino acidresidues cited in this paper refers to this amino acid sequence of themature protein.

FIG. 4. Dose-dependent apoA-I nitration and the impairment of reversecholesterol transport activity. HDL was nitrated in a series of eitherMPO/H₂O₂/NO₂ ⁻ reactions with increasing amounts of H₂O₂ orperoxynitrite, as indicated, for 1.5 h. A) The effect of the nitrationreactions on the ABCA1-dependent reverse cholesterol efflux of the HDL.The modified HDL from each reaction was subsequently incubated withmurine RAW264 macrophages loaded with ³H-cholesterol-labeled acetylatedlow density lipoprotein (AcLDL). These cells were treated with themodified HDL in the presence of cAMP to measure the ABCA1-dependentcomponent of reverse cholesterol transport. After 4 hours, media andcellular ³H was counted and the percent efflux calculated as the amountof ³H-cholesterol in the media divided by the total ³H-cholesterol(media+cellular). All values were normalized to the ABCA1-dependentcholesterol efflux obtained with unmodified HDL. B) Site-specificquantitation of apoA-I nitration determined using LC-MS. The proteins inthe modified HDL reaction were precipitated with cold acetone, separatedby SDS-PAGE, and detected by Coomassie blue staining. The apoA-I was cutfrom the gel and digested by trypsin. The progression of the nitrationreaction was followed quantitatively using the native reference peptidemethod. The peak area ratio of each tryptic peptide containing therespective tyrosines of interest to the native reference peptide wasmeasured. The percent modification of each peptide was determined basedon the decrease of the amount of each peptide relative to an untreatedcontrol.

FIG. 5. Dose-dependent apoA-I chlorination and the impairment of reversecholesterol transport activity. HDL was chlorinated in a series ofeither MPO/H₂O₂/Cl⁻ reactions with increasing amounts of H₂O₂ or HOCl,as indicated, for 1.5 h. A) The effect of the respective chlorinationreactions on the ABCA1-dependent reverse cholesterol efflux of the HDL.The efflux was determined using the same methods described in FIG. 4 forthe nitration product. B) Site-specific quantitation of apoA-Ichlorination determined using LC-MS. The methods used to detect andcharacterize these sites are given in FIG. 4.

FIG. 6. ApoA-I modification inhibits lipid binding coordinately withABCA1-dependent cholesterol acceptor activity. A) ApoA-I lipid bindingactivity was assayed by its ability to inhibit phospholipase C(PLC)-induced LDL aggregation over a 1 h time course at 37° C. 3 μg/mlof apoA-I or H₂O₂ modified apoA-I decreased LDL aggregation toapproximately the same extent. Compared to unmodified apoA-I, thecomplete MPO/H₂O₂/Cl⁻ modification system and to a lesser extent theMPO/H₂O₂/NO₂ ⁻ system led to apoA-I with decreased ability to bind tothe PLC treated LDL and inhibit its aggregation. The values representthe means of triplicate wells. The ability of apoA-I to inhibit LDLaggregation was dose dependent (data not shown). B) Lipid binding wascalculated from the initial slopes from the experiment shown in FIG. 6A,and normalized to the value for unmodified apoA-I (x-axis). Theidentical apoA-I preparations were used to assay ABCA1-dependent lipidefflux from 8Br-cAMP treated RAW264.7 in triplicate (as shown in FIG.5B), and normalized to the value for unmodified apoA-I (y-axis). MPOmodifications led to coordinate reductions in both lipid binding andABCA1-dependent cholesterol acceptor activity (linear regressionr²=0.96. p<0.0001).

FIG. 7. Detection of nitrated peptides in apoA-I isolated from humanatheroma tissue by LC-tandem mass spectrometry. Selected reactionmonitoring chromatograms (SRM) for the detection, in vivo, ofun-modified, nitrated, and chlorinated peptides, after in-gel trypticdigestion of apoA-I immunoaffinity purified from human atheroma tissue.A) The detection of the un-modified and nitrated forms of peptidecontaining the favored Y192 site determined by the in vitro experiments.B) The CID spectrum recorded at this retention time to confirm theidentity of the peptide and position of the nitration. C) The detectionof the un-modified, nitrated, and chlorinated forms (top to bottom,respectively) of the peptide containing the secondary Y166 site. Again,the CID spectra recorded at this retention time (D) confirm the identityof the peptides and the position of the nitration and chlorination. TheCID spectrum of the nitrated peptide is unambiguous while the CIDspectrum of the molecular ion of the putative chlorinated peptide alsoshows significant overlap with the trihydroxyphenylalanine-containingform of this peptide that co-elutes and has a similar molecular weight(SEQ ID NOs: 4 and 5, respectively, in order of appearance.).

FIG. 8. Detection of nitrated peptides in apoA-I isolated from humanatheroma tissue by LC-tandem mass spectrometry. Selected reactionmonitoring chromatograms (SRM) for the detection, in vivo, ofun-modified, oxidized peptides, after in-gel tryptic digestion of apoA-Iimmunoaffinity purified from human atheroma tissue. A) The CID spectrumof the oxidized peptide (T₁₆₁-R₁₇₁) of apoA-I. A residue with identicalmass to trihydroxyphenylalanine (TOPA) or tyrosine hydroperoxide isdetected at position 166 using CID spectrum. B) The CID spectrum of anun-modified peptide (D₂₈-K₄₀) of apoA-I. C). The CID spectrum of theoxidized peptide (D₂₈-K₄₀) of apoA-I. A residue with identical mass toTOPA or tyrosine hydroperoxide is detected at position 29, anddihydroxyphenylalanine is detected at position 33 (SEQ ID NOs: 12 and13, respectively, in order of appearance.).

FIG. 9. A kinetic model for myeloperoxidase.

FIG. 10. A schematic representation of certain myeloperoxidase generatedreactive intermediates and some MPO-generated oxidation products.

DETAILED DESCRIPTION

The present invention will now be described by reference to moredetailed embodiments, with occasional reference to the accompanyingdrawings. This invention may, however, be embodied in different formsand should not be construed as limited to the embodiments set forthherein. Rather these embodiments are provided so that this disclosurewill be thorough and complete, and will convey the scope of theinvention to those skilled in the art.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. The terminology used in thedescription of the invention herein is for describing particularembodiments only and is not intended to be limiting of the invention. Asused in the description of the invention and the appended claims, thesingular forms “a,” “an,” and “the” are intended to include the pluralforms as well, unless the context clearly indicates otherwise.

Unless otherwise indicated, all numbers expressing quantities ofingredients, properties such as molecular weight, reaction conditions,and so forth as used in the specification and claims are to beunderstood as being modified in all instances by the term “about.”Accordingly, unless otherwise indicated, the numerical properties setforth in the following specification and claims are approximations thatmay vary depending on the desired properties sought to be obtained inembodiments of the present invention. Notwithstanding that the numericalranges and parameters setting forth the broad scope of the invention areapproximations, the numerical values set forth in the specific examplesare reported as precisely as possible. Any numerical values, however,inherently contain certain errors necessarily resulting from error foundin their respective measurements.

All publications, patent applications, patents, and other referencesmentioned herein are incorporated by reference in their entirety.

Methods and Markers for Predicting Risk of Cardiovascular Disease

Provided herein are methods and markers for characterizing a subject'srisk for developing CVD, having CVD, or experiencing a complication ofCVD. In this context, such methods and markers are useful forcharacterizing a subject's risk of having vulnerable plaque orexperiencing a myocardial infarction.

In one embodiment, the method comprises determining levels of one ormore oxidized apoA-I-related biomolecule in a biological sample obtainedfrom the subject. In one embodiment, at least one of the oxidized apoA-Irelated biomolecules is an oxidized form of HDL. The term “high densitylipoprotein” or “HDL” as used herein is defined in accordance withcommon usage of those of skill in the art.

In another embodiment, at least one of the oxidized apoA-I-relatedbiomolecules is an oxidized form of apolipoprotein Al. In anotherembodiment, at least one of the oxidized apoA-I related biomolecules isan oxidized apoA-I peptide fragment. Such fragment is three (3) or moreamino acids in length and, except for the oxidized amino acid residuescontained therein, comprises an amino acid sequence identical to aportion of SEQ ID NO: 1. In certain embodiments such apoA-I peptidefragments comprise one or more oxidized amino acids that indicate thatthe apoA-I protein from which the peptide has been derived was oxidizedby a myeloperoxidase (“MPO”)-related system. In certain embodiments, theoxidized amino acid is at any one of positions 18, 29, 166, 192, 236, orany combination thereof, in SEQ ID NO: 1. ApoA-I oxidation may takeplace by exposure to MPO-generated reactive chlorinating species (likethose formed by the MPO/H₂O₂/Cl⁻ system, or HOCl), or MPO-relatedreactive nitrogen species (like those formed by the MPO/H₂O₂/NO₂ ⁻system, or ONOO⁻), or alternative MPO-related oxidation pathways (e.g.,MPO-generated tyrosyl radical generating systems). Thus, examples ofsuitable peptides include, but are not limited to, apoA-I peptidefragments that comprise chlorotyrosine, nitrotyrosine, dityrosine,methionine sulfoxide, oxohistidine, trihydroxyphenylalanine,dihydroxyphenylalanine, tyrosine peroxide, or other oxidized amino acidsformed by exposure of apoA-I to MPO-generated oxidants. Examples ofsuitable apoA-I peptide fragments include, but are not limited to thepeptides shown in Table 1 below.

HDL + MPO/H₂O₂/Cl⁻ Major modified peptides detected H₂O₂ (μM) byESI/MS/MS 10 25 100 L₁₈₉AEY_(Cl)HAK₁₉₅ (SEQ ID NO: 4) ♦ ♦ ♦T₁₆₁HLAPY_(Cl)SDELR₁₇₀ (SEQ ID NO: 5) ♦ ♦ D₂₈Y_(Cl)GSALGK₄₀ (SEQ ID NO:6) ♦ V₂₂₇SFLSALEEY_(Cl)TK₂₃₈ (SEQ ID NO: 7) ♦ HDL + ONOO⁻ Major modifiedpeptides detected T₁₆₁HLAPY_(NO2)SDELR₁₇₀ (SEQ ID NO: 5)D₁₃LATVY_(NO2)VDVLK₂₃ (SEQ ID NO: 8) V₂₂₇SFLSALEEY_(NO2)TK₂₃₈ (SEQ IDNO: 7)

Levels of the one or more oxidized apoA-I-related biomolecules in thebodily sample of the test subject may then be compared to a controlvalue that is derived from levels of the one or more apoA-I-relatedbiomolecules in comparable bodily samples of control subjects. In analternative embodiment, levels of the one or more oxidizedapoA-I-related biomolecules in the bodily sample of the test subject maythen be compared to an internal standard based on levels of otheroxidized biomolecules in the subject's bodily sample. Examples ofsuitable internal standard molecules include, but are not limited tolevels of oxidized total protein in the subject's bodily sample andlevels of oxidized albumin in the subject's bodily sample. Test subjectswhose levels of the one or more apoA-I-related biomolecules are abovethe control value or in the higher range of control values are atgreater risk of having or developing cardiovascular disease than testsubjects whose levels of the one more apoA-I-related biomolecules are ator below the control value or in the lower range of control values.Moreover, the extent of the difference between the subject's oxidizedapoA-I-related biomolecule levels and the control value is also usefulfor characterizing the extent of the risk and thereby, determining whichsubjects would most greatly benefit from certain therapies.

In certain embodiments, the subject's risk profile for CVD is determinedby combining a first risk value, which is obtained by comparing levelsof one or more apoA-I related biomolecules in a bodily sample of thesubject with levels of said one or more apoA-I-related biomolecules in acontrol population, with one or more additional risk values to provide afinal risk value. Such additional risk values may be obtained byprocedures including, but not limited to, determining the subject'sblood pressure, assessing the subject's response to a stress test,determining levels of myeloperoxidase, C-reactive protein, low densitylipoprotein, or cholesterol in a bodily sample from the subject, orassessing the subject's atherosclerotic plaque burden.

In one embodiment, the method is used to assess the test subject's riskof having cardiovascular disease. Medical procedures for determiningwhether a human subject has coronary artery disease or is at risk forexperiencing a complication of coronary artery disease include, but arenot limited to, coronary angiography, coronary intravascular ultrasound(IVUS), stress testing (with and without imaging), assessment of carotidintimal medial thickening, carotid ultrasound studies with or withoutimplementation of techniques of virtual histology, coronary arteryelectron beam computer tomography (EBTC), cardiac computerizedtomography (CT) scan, CT angiography, cardiac magnetic resonance imaging(MRI), and magnetic resonance angiography (MRA.). Because cardiovasculardisease, typically, is not limited to one region of a subject'svasculature, a subject who is diagnosed as having or being at risk ofhaving coronary artery disease is also considered at risk of developingor having other forms of CVD such as cerebrovascular disease,aortic-iliac disease, and peripheral artery disease. Subjects who are atrisk of having cardiovascular disease are at risk of having an abnormalstress test or abnormal cardiac catherization. Subjects who are at riskof having CVD are also at risk of exhibiting increased carotid intimalmedial thickness and coronary calcification, characteristics that can beassessed using non-invasive imaging techniques. Subjects who are at riskof having CVD are also at risk of having an increased atheroscleroticplaque burden, a characteristic that can be examined using intravascularultrasound.

In another embodiment, the present methods are used to assess the testsubject's risk of developing cardiovascular disease in the future. Inone embodiment, the test subject is an apparently healthy individual. Inanother embodiment, the subject is not otherwise at elevated risk ofhaving cardiovascular disease. In another embodiment, the presentmethods are used to determine if a subject presenting with chest pain isat risk of experiencing a heart attack or other major adverse cardiacevent, such as a heart attack, a myocardial infarction, reinfarction,the need for revascularization, or death, near term. after the subjectpresents with chest pain. As used herein, the term “near term” meanswithin one year. Thus, subjects who are at near term risk may be at riskof experiencing a major adverse cardiac event within the following day,3 months, or 6 months after presenting with chest pain.

The present invention also provides a method for monitoring over timethe status of CVD in a subject who has been diagnosed as having CVD. Inthis context, the method is also useful for monitoring the risk foratherosclerotic progression or regression in a subject with CVD. In oneembodiment, the method comprises determining the levels of one or moreof the oxidized apoA-I-related biomolecules in a biological sample takenfrom the subject at an initial time and in a corresponding biologicalsample taken from the subject at a subsequent time. An increase inlevels of the one or more oxidized apoA-I-related biomolecules in abiological sample taken at the subsequent time as compared to theinitial time indicates that the subject's CVD has progressed orworsened. A decrease in levels of the one or more oxidizedapoA-I-related molecules indicates that the CVD has improved orregressed. For those subjects who has already experienced an acuteadverse cardiovascular event such as a myocardial infarction or ischemicstroke, such method can also be used to assess the subject's risk ofhaving a subsequent acute adverse cardiovascular event. An increase overtime in levels of the one or more oxidized apoA-I-related biomoleculesin the subject indicates that a subject's risk of experiencing asubsequent adverse cardiovascular event has increased. A decrease overtime in levels of the one or more oxidized apoA-I-related biomoleculesin the subject indicates that that the subject's risk of experiencing asubsequent adverse cardiovascular event has decreased.

In another embodiment, the present invention provides a method forevaluating therapy in a subject suspected of having or diagnosed ashaving cardiovascular disease. The method comprises determining levelsof one or more oxidized apoA-I-related biomolecules, including oxidizedHDL, oxidized apoA-I, an oxidized peptide fragment of apoA-I, andcombinations thereof, in a biological sample taken from the subjectprior to therapy and determining levels of the one or more of theoxidized apoA-I related biomolecules in a corresponding biologicalsample taken from the subject during or following therapy. A decrease inlevels of the one or more oxidized apoA-I-related biomolecules in thesample taken after or during therapy as compared to levels of the one ormore oxidized apoA-I-related biomolecules in the sample taken beforetherapy is indicative of a positive effect of the therapy oncardiovascular disease in the treated subject.

The present cardiovascular disease predictive methods are based, atleast in part, on inventors' discovery that, as compared to totalproteins, apoliprotein Al in bodily samples obtained from subjects withcardiovascular disease is preferentially oxidized, e.g., nitrated orchlorinated. It is believed that the presence of these oxidized apoA-Irelated biomolecules are produced through MPO generated oxidants. MPO(donor: hydrogen peroxide, oxidoreductase, EC 1.11.1.7) is a tetrameric,heavily glycosylated, basic (PI. 10) heme protein of approximately 150kDa. It is comprised of two identical disulfide-linked protomers, eachof which possesses a protoporphyrin-containing 59-64 kDa heavy subunitand a 14 kDa light subunit (Nauseef, W. M, et al., Blood 67:1504-1507;1986.)

MPO is abundant in neutrophils and monocytes, accounting for 5%, and 1to 2%, respectively, of the dry weight of these cells (Nauseef, W. M, etal., Blood 67:1504-1507; 1986., (Hurst, J. K. In: Everse J.; Everse K.;Grisham M. B., eds. Peroxidases in chemistry and biology 1st ed. BocaRaton: CRC Press; 1991:37-62.) The heme protein is stored in primaryazurophilic granules of leukocytes and secreted into both theextracellular milieu and the phagolysosomal compartment followingphagocyte activation by a variety of agonists (Klebanoff, S. J, et al.The neutrophil: functions and clinical disorders. Amsterdam: ElsevierScientific Publishing Co.; 1978.)

A recently proposed working kinetic model for MPO is shown in FIG. 12.MPO is a complex heme protein which possesses multiple intermediatestates, each of which are influenced by the availability of reducedoxygen species such as O₂ ⁻ and H₂O₂, and nitric oxide (NO, nitrogenmonoxide) (Abu-Soud, H. M., et al., J. Biol. Chem. 275:5425-5430; 2000).At ground state, MPO exists in the ferric (Fe(III)) form. Upon additionof H₂O₂, the heme group of MPO is oxidized by two e⁻ equivalents forminga reactive ferryl π cation radical intermediate termed Compound I. Inthe presence of halides such as Cl⁻, Br⁻, and I⁻, and the psuedohalidethiocyanate (SCN⁻), Compound I is readily reduced in a single two e⁻step, regenerating MPO-Fe(III) and the corresponding hypohalous acid(HOX). At plasma levels of halides and thiocyanate (100 mM Cl⁻ 100 mMBr⁻, 50 mM SCN⁻, 100 nM I⁻, chloride is a preferred substrate andhypochlorous acid (HOCl), a potent chlorinating oxidant, is formed(Foote, C. S., et al; Nature 301:715-726; 1983., Weiss, S. J., et al. J.Clin. Invest. 70:598-607; 1982).

Compound I can also oxidize numerous organic substrates while the hemeundergoes two sequential one e⁻ reduction steps, generating compound IIand MPO-Fe(III), respectively (FIG. 12). Low molecular weight compoundsprimarily serve as substrates for MPO, generating diffusible oxidantsand free radical species which can then convey the oxidizing potentialof the heme to distant targets. In addition to halides and SCN⁻, some ofthe naturally occurring substrates for MPO include nitrite (NO₂ ⁻) (vander Vliet, A., et al., J. Biol. Chem. 272:7617-7625; 1997), tyrosine(van der Vliet, A., et al., J. Biol. Chem. 272:7617-7625; 1997),ascorbate (Marquez, L. A., et al., J. Biol. Chem. 265:5666-5670; 1990),urate (Maehly, H. C. Methods Enzymol. 2:798-801; 1955), catecholamines(Metodiewa, D., et al., Eur. J. Biochem. 193:445-448; 1990), estrogens(Klebanoff, S. J. J. Exp. Med. 145:983-998; 1977), and serotonin(Svensson, B. E. Chem. Biol. Interact. 70:305-321; 1989). MPO-Fe(III)can also be reduced to an inactive ferrous form, MPO-Fe(II) (Hurst, J.K. In: Everse J.; Everse K.; Grisham M. B., eds. Peroxidases inchemistry and biology 1st ed. Boca Raton: CRC Press; 1991:37-62,(Kettle, A. J., et al., Redox. Rep. 3:3-15; 1997). MPO-Fe(III) andMPO-Fe(II) bind to O₂ ^(•−), and O₂, respectively, forming a ferrousdioxy intermediate, compound III (MPO-Fe(II)-O₂) (FIG. 12). Spectralstudies demonstrate that addition of H₂O₂ to Compound III ultimatelyforms compound II. Thus, compound III may indirectly promote one e⁻peroxidation reactions.

Recent studies identify a role for NO, a relatively long-lived freeradical generated by nitric oxide synthase (NOS), in modulating MPOperoxidase activity (Abu-Soud, H. M., et al., J. Biol. Chem.275:5425-5430; 2000). MPO and the inducible isoform of NOS arecolocalized in the primary granule of leukocytes. During phagocyteactivation, such as during ingestion of bacteria, MPO and NOS aresecreted into the phagolysosome and extracellular compartments, andnitration of bacterial proteins is observed (Evans, T. J., et al., Proc.Natl. Acad. Sci. USA 93:9553-9558; 1996). Rapid kinetics studiesdemonstrate that at low levels of NO, the initial rate of MPO-catalyzedperoxidation of substrates is enhanced. The mechanism is throughacceleration of the rate-limiting step in MPO catalysis, reduction ofcompound II to MPO-Fe(III) (FIG. 1) (Abu-Soud, H. M., et al., J. Biol.Chem. 275:5425-5430; 2000., Abu-Soud, H. M., et al. Nitric oxide is aphysiological substrate for mammalian animal peroxidases. J. Biol. Chem.275:37524-37532, 2000). At higher levels of NO, reversible inhibition ofMPO occurs through formation of a spectroscopically distinguishablenitrosyl complex, MPO-Fe(III)-NO (Abu-Soud, H. M., et al., J. Biol.Chem. 275:5425-5430; 2000). NO also can serve as a substrate for MPOcompound I, resulting in its reduction to Compound II (Id.).Furthermore, in the presence of NO, the overall turnover rate of MPOthrough the peroxidase cycle is enhanced nearly 1000-fold (Id.).Finally, NO also reversibly binds to MPO-Fe(II) forming thecorresponding MPO-Fe(II)-NO intermediate, which is in equilibrium withMPO-Fe(II) and MPO-Fe(III)-NO (FIG. 1) (Abu-Soud, H. M., et al., J.Biol. Chem. 275:5425-5430; 2000., Abu-Soud, H. M., et al. Nitric oxideis a physiological substrate for mammalian animal peroxidases. J. Biol.Chem. 275:37524-37532, 2000).

As described above, MPO can utilize a variety of cosubstrates with H₂O₂to generate reactive oxidants as intermediates. Many stable end-productsgenerated by these species have been characterized and shown to beenriched in proteins, lipids, and LDL recovered from humanatherosclerotic lesions (Chisolm, G. M., et al., Proc. Natl. Acad. Sci.USA 91:11452-11456; 1994, Hazell, L. J., et al, J. Clin. Invest.97:1535-1544; 1996, Hazen, S. L., et al., J. Clin. Invest. 99:2075-2081;1997, Leeuwenburgh, C., et al, J. Biol. Chem. 272:1433-1436; 1997,Leeuwenburgh, C., et al., J. Biol. Chem. 272:3520-3526; 1997). FIG. 13summarizes some of the reactive intermediates and products formed byMPO, any of which are known to be enriched in vascular lesions.

Biological Samples

Suitable biological samples useful for predicting or monitoringcardiovascular disease in a subject or for assessing the effect oftherapeutic agents on subjects with cardiovascular disease include butare not limited to whole blood samples, samples of blood fractions,including but not limited to serum and plasma. The sample may be freshblood or stored blood (e.g., in a blood bank) or blood fractions. Thesample may be a blood sample expressly obtained for the assays of thisinvention or a blood sample obtained for another purpose which can besubsampled for the assays of this invention.

In one embodiment, the biological sample is whole blood. Whole blood maybe obtained from the subject using standard clinical procedures. Inanother embodiment, the biological sample is plasma. Plasma may beobtained from whole blood samples by centrifugation of anti-coagulatedblood. Such process provides a buffy coat of white cell components and asupernatant of the plasma. In another embodiment, the biological sampleis serum. Serum may be obtained by centrifugation of whole blood samplesthat have been collected in tubes that are free of anti-coagulant. Theblood is permitted to clot prior to centrifugation. Theyellowish-reddish fluid that is obtained by centrifugation is the serum.

The sample may be pretreated as necessary by dilution in an appropriatebuffer solution, heparinized, concentrated if desired, or fractionatedby any number of methods including but not limited toultracentrifugation, fractionation by fast performance liquidchromatography (FPLC), or precipitation of apolipoprotein B containingproteins with dextran sulfate or other methods. Any of a number ofstandard aqueous buffer solutions, employing one of a variety ofbuffers, such as phosphate, Tris, or the like, at physiological pH canbe used.

Subjects

The subject is any human or other animal to be tested for characterizingits risk of CVD. In certain embodiments, the subject does not otherwisehave an elevated risk of an adverse cardiovascular event. Subjectshaving an elevated risk of an adverse cardiovascular event include thosewith a family history of cardiovascular disease, elevated lipids,smokers, prior acute cardiovascular event, etc. (See, e.g., Harrison'sPrinciples of Experimental Medicine, 15th Edition, McGraw-Hill, Inc.,N.Y. —hereinafter “Harrison's”).

In certain embodiments the subject is an apparently healthy nonsmoker.“Apparently healthy”, as used herein, means individuals who have notpreviously being diagnosed as having any signs or symptoms indicatingthe presence of atherosclerosis, such as angina pectoris, history of anacute adverse cardiovascular event such as a myocardial infarction orstroke, evidence of atherosclerosis by diagnostic imaging methodsincluding, but not limited to coronary angiography. Apparently healthyindividuals also do not otherwise exhibit symptoms of disease. In otherwords, such individuals, if examined by a medical professional, would becharacterized as healthy and free of symptoms of disease. “Nonsmoker”means an individual who, at the time of the evaluation, is not a smoker.This includes individuals who have never smoked as well as individualswho in the past have smoked but presently no longer smoke.

Immunoassays for Determining Levels of Oxidized HDL, apoA-I and apoA-IPeptide Fragments

Levels of the oxidized HDL, apoA-I, and apoA-I peptide fragments in thebiological sample can be determined using polyclonal or monoclonalantibodies that are immunoreactive with such oxidized biomolecule. Forexample, antibodies immunospecific for nitrotyrosine containing apoA-Ipeptide fragments may be made and labeled using standard procedures andthen employed in immunoassays to detect the presence of suchnitrotyrosine containing apoA-I peptide in the sample. Suitableimmunoassays include, by way of example, radioimmunoassays, both solidand liquid phase, fluorescence-linked assays, competitive immunoassays,or enzyme-linked immunosorbent assays. In certain embodiments, theimmunoassays are also used to quantify the amount of the oxidizedbiomolecule that is present in the sample.

Monoclonal antibodies raised against the select oxidized polypeptidespecies are produced according to established procedures. Generally, theoxidized apoA-I protein or apoA-I peptide fragment is used to immunize ahost animal.

Suitable host animals, include, but are not limited to, rabbits, mice,rats, goats, and guinea pigs. Various adjuvants may be used to increasethe immunological response in the host animal. The adjuvant useddepends, at least in part, on the host species. Such animals produceheterogenous populations of antibody molecules, which are referred to aspolyclonal antibodies and which may be derived from the sera of theimmunized animals.

Monoclonal antibodies, which are homogenous populations of an antibodythat bind to a particular antigen, are obtained from continuous cellslines. Conventional techniques for producing monoclonal antibodies arethe hybridoma technique of Kohler and Millstein (Nature 356:495-497(1975)) and the human B-cell hybridoma technique of Kosbor et al(Immunology Today 4:72 (1983)). Such antibodies may be of anyimmunoglobulin class including IgG, IgM, IgE, Iga, IgD and any classthereof. Procedures for preparing antibodies against modified aminoacids, such as for example, 3-nitrotyrosine are described in Ye, Y. Z.,M. Strong, Z. Q. Huang, and J. S. Beckman. 1996. Antibodies thatrecognize nitrotyrosine. Methods Enzymol. 269:201-209.

Preparation of Antibodies

The oxidized apoA-I protein or oxidized apoA-I peptide fragment can beused as an immunogen to produce antibodies immunospecific for theoxidized protein or peptide fragment. The term “immunospecific” meansthe antibodies have substantially greater affinity for the oxidizedapoA-I protein or apoA-I peptide fragment than for other proteins orpolypeptides, including the un-oxidized apoA-I protein or apoA-I peptidefragment. Such antibodies may include, but are not limited to,polyclonal, monoclonal, chimeric, single chain, and Fab fragments.

The oxidized apoA-I peptide fragments are at least three amino acids inlength and comprise a modified apoA-I protein sequence, i.e., thepeptide comprises a sequence that, except for the presence of anoxidized amino acid, particularly an oxidized tyrosine residue, isidentical to a sequence in SEQ ID NO: 1. The apoA-I peptide fragmentscan be 3 amino acids in length. In other embodiments, the apoA-I peptidefragment is 4, 5, 6, 7, 8, 9, or 10 amino acids in length. In otherembodiments, the apoA-I peptide fragment is 11-20, 21-30, 31-40, 41-50,51-60, 61-70, 71-80, 81-90, 91-100, 101-110, 111-120, 121-130, 131-140,141-150, 151-160, 161-170, 171-180, 181-190, 191-200, 201-210, 211-220,221-230, or 231-242 amino acids in length.

Peptides that are less than 6 amino acids in length conventionally arefused with those of another protein such as keyhole limpet hemocyaninand antibody chimeric molecule. Larger fragments, e.g., apoA-I peptidefragments that are from 6 to 242 amino acids in length may also be usedas the immunogen. The structure of larger immunogenic fragments of theapoA-I protein can be determined using software programs, for examplethe MacVector program, to determine hydrophilicity and hydrophobicityand ascertain regions of the protein that are likely to be present atthe surface of the molecule.

Polyclonal antibodies are generated using conventional techniques byadministering the apoA-I protein or apoA-I peptide fragment. or apoA-Ito a host animal. Depending on the host species, various adjuvants maybe used to increase immunological response. Among adjuvants used inhumans, Bacilli-Calmette-Guerin (BCG), and Corynebacterium parvum. areespecially preferable. Conventional protocols are also used to collectblood from the immunized animals and to isolate the serum and or the IgGfraction from the blood.

For preparation of monoclonal antibodies, conventional hybridomatechniques are used. Such antibodies are produced by continuous celllines in culture. Suitable techniques for preparing monoclonalantibodies include, but are not limited to, the hybridoma technique, thehuman B-cell hybridoma technique, and the EBV hybridoma technique.

Various immunoassays may be used for screening to identify antibodieshaving the desired specificity. These include protocols that involvecompetitive binding or immunoradiometric assays and typically involvethe measurement of complex formation between the respective oxidizedapoA-I polypeptide and the antibody.

The present antibodies may be used to detect the presence of or measurethe amount of oxidized HDL, oxidized apoA-I, and oxidized apoA-I peptidefragments in a biological sample from the subject. The method comprisescontacting a sample taken from the individual with one or more of thepresent antibodies; and assaying for the formation of a complex betweenthe antibody and a protein or peptide in the sample. For ease ofdetection, the antibody can be attached to a substrate such as a column,plastic dish, matrix, or membrane, preferably nitrocellulose. The samplemay be a tissue or a biological fluid, including urine, whole blood, orexudate, preferably serum. The sample may be untreated, subjected toprecipitation, fractionation, separation, or purification beforecombining with the antibody. Interactions between antibodies in thesample and the isolated HDL, protein or peptide are detected byradiometric, colorimetric, or fluorometric means, size-separation, orprecipitation. Preferably, detection of the antibody-protein or peptidecomplex is by addition of a secondary antibody that is coupled to adetectable tag, such as for example, an enzyme, fluorophore, orchromophore. Formation of the complex is indicative of the presence ofoxidized HDL, apoA-I or apoA-I peptide fragments in the individual'sbiological sample.

In certain embodiments, the method employs an enzyme-linkedimmunosorbent assay (ELISA) or a Western immunoblot procedure.

Additional Methods for Measuring of Oxidized HDL, Oxidized apoA-I andOxidized apoA-I Peptide Fragments

Mass spectrometry-based methods (e.g. LC/ESI/MS/MS) may also be used toassess levels of oxidized HDL, oxidized apoA-I and oxidized apoA-Ipeptide fragments in the biological sample as shown in the examplesbelow. Such methods are standard in the art and include, for example,HPLC with on-line electrospray ionization tandem mass spectometry.Synthetic standard tryptic digets peptides for parent (unmodified) andmodified (nitrated, chlorinated) forms can be made readily withautomated peptide synthesizers using commercially available fmocmodified amino acids. The parent molecules i.e., the unmodified HDL,apoA-I, and apoA-I peptide fragments will have different masses than theoxidized molecules because of added moieties, added NO₂ or moiety, forexample). Thus, distinct parent->daughter ion transitions for eachpeptide would be achievable. Adding the nitro group to Tyr changes thepKa of the phenoxy hydrogen on the Tyr from 10 to 7. Thus, chargedifferences and changes in polarity between modified and non-modifiedpeptide have high likelihood of showing distinct retention times on HPLCas well.

Control Value

Levels of the oxidized HDL, apoA-I and/or apoA-I polypeptide in thebiological sample obtained from the test subject may compared to acontrol value. The control value is based upon levels of oxidized HDL,apoA-I, and/or apoA-I, respectively, in comparable samples obtained froma control population, e.g., the general population or a selectpopulation of human subjects. For example, the select population may becomprised of apparently healthy subjects. “Apparently healthy”, as usedherein, means individuals who have not previously had any signs orsymptoms indicating the presence of atherosclerosis, such as anginapectoris, history of an acute adverse cardiovascular event such as amyocardial infarction or stroke, evidence of atherosclerosis bydiagnostic imaging methods including, but not limited to coronaryangiography. Apparently healthy individuals also do not otherwiseexhibit symptoms of disease. In other words, such individuals, ifexamined by a medical professional, would be characterized as healthyand free of symptoms of disease. In another example, the control valuecan be derived from an apparently healthy nonsmoker population.“Nonsmoker,” as used herein, means an individual who, at the time of theevaluation, is not a smoker. This includes individuals who have neversmoked as well as individuals who in the past have smoked but presentlyno longer smoke. An apparently healthy, nonsmoker population may have adifferent normal range of oxidized HDL, apoA-I and/or apoA-I peptidefragment than will a smoking population or a population whose memberhave had a prior cardiovascular disorder. Accordingly, the controlvalues selected may take into account the category into which the testsubject falls. Appropriate categories can be selected with no more thanroutine experimentation by those of ordinary skill in the art.

The control value is related to the value used to characterize the levelof the oxidized polypeptide obtained from the test subject. Thus, if thelevel of the oxidized polypeptide is an absolute value such as the unitsof oxidized apoA-I per ml of blood, the control value is also based uponthe units of oxidized apoA-I per ml of blood in individuals in thegeneral population or a select population of human subjects. Similarly,if the level of the oxidized HDL, apoA-I, or apoA-I peptide fragment isa representative value such as an arbitrary unit obtained from acytogram, the control value is also based on the representative value.

The control value can take a variety of forms. The control value can bea single cut-off value, such as a median or mean. The control value canbe established based upon comparative groups such as where the risk inone defined group is double the risk in another defined group. Thecontrol values can be divided equally (or unequally) into groups, suchas a low risk group, a medium risk group and a high-risk group, or intoquadrants, the lowest quadrant being individuals with the lowest riskthe highest quadrant being individuals with the highest risk, and thetest subject's risk of having CVD can be based upon which group his orher test value falls.

Control values of oxidized HDL, apoA-I and/or apoA-I peptide fragment inbiological samples obtained, such as for example, mean levels, medianlevels, or “cut-off” levels, are established by assaying a large sampleof individuals in the general population or the select population andusing a statistical model such as the predictive value method forselecting a positivity criterion or receiver operator characteristiccurve that defines optimum specificity (highest true negative rate) andsensitivity (highest true positive rate) as described in Knapp, R. G.,and Miller, M. C. (1992). Clinical Epidemiology and Biostatistics.William and Wilkins, Harual Publishing Co. Malvern, Pa., which isspecifically incorporated herein by reference. A “cutoff” value can bedetermined for each risk predictor that is assayed. The standardizedmethod that was used in Example 1 below employs the guaiacol oxidationassay as described in Klebanoff, S. J., Waltersdorph, A. N. and Rosen,H. 1984. “Antimicrobial activity of myeloperoxidase”. Methods inEnzymology. 105: 399-403).

Comparison of Oxidized Biomolecule from the Test Subject to the ControlValue

Levels of each select oxidized biomolecule, i.e., oxidized HDL, oxidizedapoA-I, oxidized apoA-I polypeptide fragment in the individual'sbiological sample may be compared to a single control value or to arange of control values. If the level of the present risk predictor inthe test subject's biological sample is greater than the control valueor exceeds or is in the upper range of control values, the test subjectis at greater risk of developing or having CVD than individuals withlevels comparable to or below the control value or in the lower range ofcontrol values. In contrast, if levels of the present risk predictor inthe test subject's biological sample is below the control value or is inthe lower range of control values, the test subject is at a lower riskof developing or having CVD than individuals whose levels are comparableto or above the control value or exceeding or in the upper range ofcontrol values. The extent of the difference between the test subject'srisk predictor levels and control value is also useful forcharacterizing the extent of the risk and thereby, determining whichindividuals would most greatly benefit from certain aggressivetherapies. In those cases, where the control value ranges are dividedinto a plurality of groups, such as the control value ranges forindividuals at high risk, average risk, and low risk, the comparisoninvolves determining into which group the test subject's level of therelevant risk predictor falls.

Alternatively, the level of oxidized biomolecule, i.e., oxidized HDL,oxidized apoA-I, or oxidized apoA-I peptide fragment may be compared tothe level of an oxidized internal standard in the sample. Examples ofsuitable internal standards include, but are not limited to, levels ofoxidized total protein in the sample or levels of oxidized albumin inthe sample.

The present predictive tests are useful for determining if and whentherapeutic agents that are targeted at preventing CVD or for slowingthe progression of CVD should and should not be prescribed for aindividual. For example, individuals with values of oxidized apoA-Iabove a certain cutoff value, or that are in the higher tertile orquartile of a “normal range,” could be identified as those in need ofmore aggressive intervention with lipid lowering agents, life stylechanges, etc.

Evaluation of CVD Therapeutic Agents

Also provided are methods for evaluating the effect of CVD therapeuticagents on individuals who have been diagnosed as having or as being atrisk of developing CVD. Such therapeutic agents include, but are notlimited to, anti-inflammatory agents, insulin sensitizing agents,antihypertensive agents, anti-thrombotic agents, anti-platelet agents,fibrinolytic agents, lipid reducing agents, direct thrombin inhibitors,ACAT inhibitor, CDTP inhibitor thioglytizone, glycoprotein II b/IIIareceptor inhibitors, agents directed at raising or altering HDLmetabolism such as apoA-I milano or CETP inhibitors (e.g., torcetrapib),or agents designed to act as artificial HDL. Such evaluation comprisesdetermining the levels of one or more oxidized apoA-I-relatedbiomolecules in a biological sample taken from the subject prior toadministration of the therapeutic agent and a corresponding biologicalfluid taken from the subject following administration of the therapeuticagent. A decrease in the level of the selected risk markers in thesample taken after administration of the therapeutic as compared to thelevel of the selected risk markers in the sample taken beforeadministration of the therapeutic agent is indicative of a positiveeffect of the therapeutic agent on cardiovascular disease in the treatedsubject.

Kits

Also provided are kits for practicing the present methods. Such kitscontain reagents for assessing levels of oxidized apoA-I, oxidizedapoA-I peptide fragments, oxidized HDL, or combinations thereof in abiological sample. In one embodiment, the reagent is an antibody that isimmunospecific for oxidized apoA-I, or an oxidized apoA-I peptidefragment, or both. In one embodiment, the kit also comprisesinstructions for using the reagent in the present methods. In anotherembodiment, the kit comprises information useful for determining asubject's risk of cardiovascular disease or a complication. Examples ofsuch information include, but are not limited cut-off values,sensitivities at particular cut-off values, as well as other printedmaterial for characterizing risk based upon the outcome of the assay. Insome embodiments, such kits may also comprise control reagents, e.g.oxidized HDL, oxidized apoA-I, and/or oxidized apoA-I peptide fragments.

Therapeutic Methods

The present invention also relates to methods of treating a subject toreduce the risk of a cardiovascular disorder or complication of suchdisorder. In one embodiment, the method comprises determining levels ofone or more apoA-I related biomolecules in a bodily sample of thesubject, and where the levels of the one or more apoA-I relatedbiomolecules are elevated as compared to levels in comparable bodilysamples from a control population of subjects, administering to thesubject an agent chosen from an anti-inflammatory agent, anantithrombotic agent, an anti-platelet agent, a fibrinolytic agent, alipid reducing agent, a direct thrombin inhibitor, a glycoproteinIIb/IIIa receptor inhibitor, an agent that binds to cellular adhesionmolecules and inhibits the ability of white blood cells to attach tosuch molecules, a calcium channel blocker, a beta-adrenergic receptorblocker, a cyclooxygenase-2 inhibitor, an angiotensin system inhibitor,and/or combinations thereof. The agent is administered in an amounteffective to lower the risk of the subject developing a futurecardiovascular disorder.

“Anti-inflammatory” agents include but are not limited to, Alclofenac;Alclometasone Dipropionate; Algestone Acetonide; Alpha Amylase;Amcinafal; Amcinafide; Amfenac Sodium; Amiprilose Hydrochloride;Anakinra; Anirolac; Anitrazafen; Apazone; Balsalazide Disodium;Bendazac; Benoxaprofen; Benzydamine Hydrochloride; Bromelains;Broperamole; Budesonide; Carprofen; Cicloprofen; Cintazone; Cliprofen;Clobetasol Propionate; Clobetasone Butyrate; Clopirac; CloticasonePropionate; Cormethasone Acetate; Cortodoxone; Deflazacort; Desonide;Desoximetasone; Dexamethasone Dipropionate; Diclofenac Potassium;Diclofenac Sodium; Diflorasone Diacetate; Diflumidone Sodium;Diflunisal; Difluprednate; Diftalone; Dimethyl Sulfoxide; Drocinonide;Endrysone; Enlimomab; Enolicam Sodium; Epirizole; Etodolac; Etofenamate;Felbinac; Fenamole; Fenbufen; Fenclofenac; Fenclorac; Fendosal;Fenpipalone; Fentiazac; Flazalone; Fluazacort; Flufenamic Acid;Flumizole; Flunisolide Acetate; Flunixin; Flunixin Meglumine; FluocortinButyl; Fluorometholone Acetate; Fluquazone; Flurbiprofen; Fluretofen;Fluticasone Propionate; Furaprofen; Furobufen; Halcinonide; HalobetasolPropionate; Halopredone Acetate; Ibufenac; Ibuprofen; IbuprofenAluminum; Ibuprofen Piconol; Ilonidap; Indomethacin; IndomethacinSodium; Indoprofen; Indoxole; Intrazole; Isoflupredone Acetate;Isoxepac; Isoxicam; Ketoprofen; Lofemizole Hydrochloride; Lornoxicam;Loteprednol Etabonate; Meclofenamate Sodium; Meclofenamic Acid;Meclorisone Dibutyrate; Mefenamic Acid; Mesalamine; Meseclazone;Methylprednisolone Suleptanate; Morniflumate; Nabumetone; Naproxen;Naproxen Sodium; Naproxol; Nimazone; Olsalazine Sodium; Orgotein;Orpanoxin; Oxaprozin; Oxyphenbutazone; Paranyline Hydrochloride;Pentosan Polysulfate Sodium; Phenbutazone Sodium Glycerate; Pirfenidone;Piroxicam; Piroxicam Cinnamate; Piroxicam Olamine; Pirprofen;Prednazate; Prifelone; Prodolic Acid; Proquazone; Proxazole; ProxazoleCitrate; Rimexolone; Romazarit; Salcolex; Salnacedin; Salsalate;Salycilates; Sanguinarium Chloride; Seclazone; Sermetacin; Sudoxicam;Sulindac; Suprofen; Talmetacin; Talniflumate; Talosalate; Tebufelone;Tenidap; Tenidap Sodium; Tenoxicam; Tesicam; Tesimide; Tetrydamine;Tiopinac; Tixocortol Pivalate; Tolmetin; Tolmetin Sodium; Triclonide;Triflumidate; Zidometacin; Glucocorticoids; Zomepirac Sodium.

“Anti-thrombotic” and/or “fibrinolytic” agents include but are notlimited to, Plasminogen (to plasmin via interactions of prekallikrein,kininogens, Factors XII, XIIIa, plasminogen proactivator, and tissueplasminogen activator[TPA]) Streptokinase; Urokinase: AnisoylatedPlasminogen-Streptokinase Activator Complex; Pro-Urokinase; (Pro-UK);rTPA (alteplase or activase; r denotes recombinant); rPro-UK;Abbokinase; Eminase; Sreptase Anagrelide Hydrochloride; Bivalirudin;Dalteparin Sodium; Danaparoid Sodium; Dazoxiben Hydrochloride; EfegatranSulfate; Enoxaparin Sodium; Ifetroban; Ifetroban Sodium; TinzaparinSodium; retaplase; Trifenagrel; Warfarin; Dextrans.

“Anti-platelet” agents include but are not limited to, Clopridogrel;Sulfinpyrazone; Aspirin; Dipyridamole; Clofibrate; Pyridinol Carbamate;PGE; Glucagon; Antiserotonin drugs; Caffeine; Theophyllin Pentoxifyllin;Ticlopidine; Anagrelide.

“Lipid-reducing” agents include but are not limited to, gemfibrozil,cholystyramine, colestipol, nicotinic acid, probucol lovastatin,fluvastatin, simvastatin, atorvastatin, pravastatin, cerivastatin, andother HMG-CoA reductase inhibitors.

“Direct thrombin inhibitors” include but are not limited to, hirudin,hirugen, hirulog, agatroban, PPACK, thrombin aptamers.

“Glycoprotein IIb/IIIa receptor inhibitors” are both antibodies andnon-antibodies, and include but are not limited to ReoPro (abcixamab),lamifiban, tirofiban.

“Calcium channel blockers” are a chemically diverse class of compoundshaving important therapeutic value in the control of a variety ofdiseases including several cardiovascular disorders, such ashypertension, angina, and cardiac arrhythmias (Fleckenstein, Cir. Res.v. 52, (suppl. 1), p. 13-16 (1983); Fleckenstein, Experimental Facts andTherapeutic Prospects, John Wiley, New York (1983); McCall, D., CurrPract Cardiol, v. 10, p. 1-11 (1985)). Calcium channel blockers are aheterogenous group of drugs that prevent or slow the entry of calciuminto cells by regulating cellular calcium channels. (Remington, TheScience and Practice of Pharmacy, Nineteenth Edition, Mack PublishingCompany, Eaton, Pa., p. 963 (1995)). Most of the currently availablecalcium channel blockers, and useful according to the present invention,belong to one of three major chemical groups of drugs, thedihydropyridines, such as nifedipine, the phenyl alkyl amines, such asverapamil, and the benzothiazepines, such as diltiazem. Other calciumchannel blockers useful according to the invention, include, but are notlimited to, amrinone, amlodipine, bencyclane, felodipine, fendiline,flunarizine, isradipine, nicardipine, nimodipine, perhexilene,gallopamil, tiapamil and tiapamil analogues (such as 1993RO-11-2933),phenyloin, barbiturates, and the peptides dynorphin, omega-conotoxin,and omega-agatoxin, and the like and/or pharmaceutically acceptablesalts thereof.

“Beta-adrenergic receptor blocking agents” are a class of drugs thatantagonize the cardiovascular effects of catecholamines in anginapectoris, hypertension, and cardiac arrhythmias. Beta-adrenergicreceptor blockers include, but are not limited to, atenolol, acebutolol,alprenolol, befunolol, betaxolol, bunitrolol, carteolol, celiprolol,hydroxalol, indenolol, labetalol, levobunolol, mepindolol, methypranol,metindol, metoprolol, metrizoranolol, oxprenolol, pindolol, propranolol,practolol, practolol, sotalolnadolol, tiprenolol, tomalolol, timolol,bupranolol, penbutolol, trimepranol,2-(3-(1,1-dimethylethyl)-amino-2-hydroxypropoxy)-3-pyridenecarbonitrilHCl,1-butylamino-3-(2,5-dichlorophenoxy-)-2-propanol,1-isopropylamino-3-(4-(2-cyclopropylmethoxyethyl)phenoxy)-2-propanol,3-isopropylamino-1-(7-methylindan-4-yloxy)-2-butanol,2-(3-t-butylamino-2-hydroxy-propylthio)-4-(5-carbamoyl-2-thienyl)thiazol,7-(2-hydroxy-3-t-butylaminpropoxy)phthalide. The above-identifiedcompounds can be used as isomeric mixtures, or in their respectivelevorotating or dextrorotating form.

Suitable COX-2 inhibitors include, but are not limited to, COX-2inhibitors described in U.S. Pat. No. 5,474,995 “Phenyl heterocycles ascox-2 inhibitors”; U.S. Pat. No. 5,521,213 “Diaryl bicyclic heterocyclesas inhibitors of cyclooxygenase-2”; U.S. Pat. No. 5,536,752 “Phenylheterocycles as COX-2 inhibitors”; U.S. Pat. No. 5,550,142 “Phenylheterocycles as COX-2 inhibitors”; U.S. Pat. No. 5,552,422 “Arylsubstituted 5,5 fused aromatic nitrogen compounds as anti-inflammatoryagents”; U.S. Pat. No. 5,604,253 “N-benzylindol-3-yl propanoic acidderivatives as cyclooxygenase inhibitors”; U.S. Pat. No. 5,604,260“5-methanesulfonamido-1-indanones as an inhibitor of cyclooxygenase-2”;U.S. Pat. No. 5,639,780 N-benzyl indol-3-yl butanoic acid derivatives ascyclooxygenase inhibitors”; U.S. Pat. No. 5,677,318Diphenyl-1,2-3-thiadiazoles as anti-inflammatory agents”; U.S. Pat. No.5,691,374 “Diaryl-5-oxygenated-2-(5H)-furanones as COX-2 inhibitors”;U.S. Pat. No. 5,698,584 “3,4-diaryl-2-hydroxy-2,5-d-ihydrofurans asprodrugs to COX-2 inhibitors”; U.S. Pat. No. 5,710,140 “Phenylheterocycles as COX-2 inhibitors”; U.S. Pat. No. 5,733,909 “Diphenylstilbenes as prodrugs to COX-2 inhibitors”; U.S. Pat. No. 5,789,413“Alkylated styrenes as prodrugs to COX-2 inhibitors”; U.S. Pat. No.5,817,700 “Bisaryl cyclobutenes derivatives as cyclooxygenaseinhibitors”; U.S. Pat. No. 5,849,943 “Stilbene derivatives useful ascyclooxygenase-2 inhibitors”; U.S. Pat. No. 5,861,419 “Substitutedpyridines as selective cyclooxygenase-2 inhibitors”; U.S. Pat. No.5,922,742 “Pyridinyl-2-cyclopenten-1-ones as selective cyclooxygenase-2inhibitors”; U.S. Pat. No. 5,925,631 “Alkylated styrenes as prodrugs toCOX-2 inhibitors”; all of which are commonly assigned to Merck FrosstCanada, Inc. (Kirkland, Calif.). Additional COX-2 inhibitors are alsodescribed in U.S. Pat. No. 5,643,933, assigned to G. D. Searle & Co.(Skokie, Ill.), entitled: “Substituted sulfonylphenylheterocycles ascyclooxygenase-2 and 5-lipoxygenase inhibitors.”

An “angiotensin system inhibitor” is an agent that interferes with thefunction, synthesis or catabolism of angiotensin II. These agentsinclude, but are not limited to, angiotensin-converting enzyme (ACE)inhibitors, angiotensin II antagonists, angiotensin II receptorantagonists, agents that activate the catabolism of angiotensin II, andagents that prevent the synthesis of angiotensin I from whichangiotensin II is ultimately derived. The renin-angiotensin system isinvolved in the regulation of hemodynamics and water and electrolytebalance. Factors that lower blood volume, renal perfusion pressure, orthe concentration of Na.sup.+ in plasma tend to activate the system,while factors that increase these parameters tend to suppress itsfunction.

Angiotensin (renin-angiotensin) system inhibitors are compounds that actto interfere with the production of angiotensin II from angiotensinogenor angiotensin I or interfere with the activity of angiotensin II. Suchinhibitors are well known to those of ordinary skill in the art andinclude compounds that act to inhibit the enzymes involved in theultimate production of angiotensin II, including renin and ACE. Theyalso include compounds that interfere with the activity of angiotensinII, once produced. Examples of classes of such compounds includeantibodies (e.g., to renin), amino acids and analogs thereof (includingthose conjugated to larger molecules), peptides (including peptideanalogs of angiotensin and angiotensin I), pro-renin related analogs,etc. Among the most potent and useful renin-angiotensin systeminhibitors are renin inhibitors, ACE inhibitors, and angiotensin IIantagonists.

Examples of angiotensin II antagonists include: peptidic compounds(e.g., saralasin, [(San¹)(Val⁵)(Ala⁸)] angiotensin-(1-8) octapeptide andrelated analogs); N-substituted imidazole-2-one (U.S. Pat. No.5,087,634); imidazole acetate derivatives including2-N-butyl-4-chloro-1-(2-chlorobenzile) imidazole-5-acetic acid (see Longet al., J. Pharmacol. Exp. Ther. 247(1), 1-7 (1988));4,5,6,7-tetrahydro-1H-imidazo[4,5-c]pyridine-6-carboxylic acid andanalog derivatives (U.S. Pat. No. 4,816,463); N2-tetrazolebeta-glucuronide analogs (U.S. Pat. No. 5,085,992); substitutedpyrroles, pyrazoles, and tryazoles (U.S. Pat. No. 5,081,127); phenol andheterocyclic derivatives such as 1,3-imidazoles (U.S. Pat. No.5,073,566); imidazo-fused 7-member ring heterocycles (U.S. Pat. No.5,064,825); peptides (e.g., U.S. Pat. No. 4,772,684); antibodies toangiotensin II (e.g., U.S. Pat. No. 4,302,386); and aralkyl imidazolecompounds such as biphenyl-methyl substituted imidazoles (e.g., EPNumber 253,310, Jan. 20, 1988); ES8891(N-morpholinoacetyl-(-1-naphthyl)-L-alanyl-(4, thiazolyl)-L-alanyl (35,45)-4-amino-3-hydroxy-5-cyclo-hexapentanoyl-N-hexylamide, SankyoCompany, Ltd., Tokyo, Japan); SKF108566 (E-alpha-2-[2-butyl-1-(carboxyphenyl)methyl]1H-imidazole-5-yl[methylane]-2-thiophenepropanoic acid,Smith Kline Beecham Pharmaceuticals, Pa.); Losartan (DUP7531MK954,DuPont Merck Pharmaceutical Company); Remikirin (RO42-5892, F. HoffmanLaRoche AG); A.sub.2 agonists (Marion Merrill Dow) and certainnon-peptide heterocycles (G. D. Searle and Company). Classes ofcompounds known to be useful as ACE inhibitors include acylmercapto andmercaptoalkanoyl prolines such as captopril (U.S. Pat. No. 4,105,776)and zofenopril (U.S. Pat. No. 4,316,906), carboxyalkyl dipeptides suchas enalapril (U.S. Pat. No. 4,374,829), lisinopril (U.S. Pat. No.4,374,829), quinapril (U.S. Pat. No. 4,344,949), ramipril (U.S. Pat. No.4,587,258), and perindopril (U.S. Pat. No. 4,508,729), carboxyalkyldipeptide mimics such as cilazapril (U.S. Pat. No. 4,512,924) andbenazapril (U.S. Pat. No. 4,410,520), phosphinylalkanoyl prolines suchas fosinopril (U.S. Pat. No. 4,337,201) and trandolopril.

Examples of renin inhibitors that are the subject of United Statespatents are as follows: urea derivatives of peptides (U.S. Pat. No.5,116,835); amino acids connected by nonpeptide bonds (U.S. Pat. No.5,114,937); di and tri peptide derivatives (U.S. Pat. No. 5,106,835);amino acids and derivatives thereof (U.S. Pat. Nos. 5,104,869 and5,095,119); diol sulfonamides and sulfinyls (U.S. Pat. No. 5,098,924);modified peptides (U.S. Pat. No. 5,095,006); peptidyl beta-aminoacylaminodiol carbamates (U.S. Pat. No. 5,089,471); pyrolimidazolones (U.S.Pat. No. 5,075,451); fluorine and chlorine statine or statone containingpeptides (U.S. Pat. No. 5,066,643); peptidyl amino diols (U.S. Pat. Nos.5,063,208 and 4,845,079); N-morpholino derivatives (U.S. Pat. No.5,055,466); pepstatin derivatives (U.S. Pat. No. 4,980,283);N-heterocyclic alcohols (U.S. Pat. No. 4,885,292); monoclonal antibodiesto renin (U.S. Pat. No. 4,780,401); and a variety of other peptides andanalogs thereof (U.S. Pat. Nos. 5,071,837, 5,064,965, 5,063,207,5,036,054, 5,036,053, 5,034,512, and 4,894,437).

In certain embodiments, the test subjects are apparently healthysubjects otherwise free of current need for treatment with the agentprescribed according to the present invention. For example, if treatmentwith a particular agent occurs based on elevated levels of oxidizedapoA-I related biomolecules, then the patient is free of symptomscalling for treatment with that agent (or the category of agent intowhich the agent falls), other than the symptom of having elevated levelsof apoA-I related biomolecules. In some embodiments, the subject isotherwise free of symptoms calling for treatment with any one of anycombination of or all of the foregoing categories of agents. Forexample, with respect to anti-inflammatory agents, the subject is freeof symptoms of rheumatoid arthritis, chronic back pain, autoimmunediseases, vascular diseases, viral diseases, malignancies, and the like.In another embodiment, the subject is not at an elevated risk of anadverse cardiovascular event (e.g., subject with no family history ofsuch events, subjects who are nonsmokers, subjects who arenon-hyperlipidemic, subjects who do not have elevated levels of asystemic inflammatory marker), other than having an elevated level ofone or more oxidized apoA-I arelated biomolecules.

In some embodiments, the subject is a non-hyperlipidemic subject. A“non-hyperlipidemic” is a subject that is a non-hypercholesterolemicand/or a non-hypertriglyceridemic subject. A “non-hypercholesterolemic”subject is one that does not fit the current criteria established for ahypercholesterolemic subject. A non-hypertriglyceridemic subject is onethat does not fit the current criteria established for ahypertriglyceridemic subject (See, e.g., Harrison's Principles ofExperimental Medicine, 15th Edition, McGraw-Hill, Inc., N.Y.—hereinafter “Harrison's”). Hypercholesterolemic subjects andhypertriglyceridemic subjects are associated with increased incidence ofpremature coronary heart disease. A hypercholesterolemic subject has anLDL level of >160 mg/dL, or >130 mg/dL and at least two risk factorsselected from the group consisting of male gender, family history ofpremature coronary heart disease, cigarette smoking (more than 10 perday), hypertension, low HDL (<35 mg/dL), diabetes mellitus,hyperinsulinemia, abdominal obesity, high lipoprotein (a), and personalhistory of cerebrovascular disease or occlusive peripheral vasculardisease. A hypertriglyceridemic subject has a triglyceride (TG) levelof >250 mg/dL. Thus, a non-hyperlipidemic subject is defined as onewhose cholesterol and triglyceride levels are below the limits set asdescribed above for both the hypercholesterolemic andhypertriglyceridemic subjects.

EXAMPLES A. Protein Studies Materials

L-[¹³C₆]tyrosine and L-[¹³C₉, ¹⁵N₁]tyrosine were purchased fromCambridge Isotopes Inc. (Andover, Mass.). Tissue culture media andadditives were purchased from Life Technologies (Gaitherburg, Md.).RAW264.7 cells were obtained from the American Type Culture Collection(Rockville, Md.). [³H]Cholesterol was obtained from Amersham(Piscataway, N.J.), and resuspended in ethanol prior to use. All otherreagents were obtained from Sigma Chemical Co. (St. Louis, Mo.) unlessotherwise specified.

Methods

General procedures. Peroxynitrite was synthesized and quantified asdescribed (Beckman, J. S., Chen, J., Ischiropoulos, H., and Crow, J. P.1994. Oxidative chemistry of peroxynitrite. Methods in Enzymology233:229-240). L-3-[¹³C₆]nitrotyrosine was synthesized fromL-[¹³C₆]tyrosine and peroxynitrite, and isolated by reverse phase HPLCto remove residual NO₂ ⁻ prior to use (Wu, W., Chen, Y., and Hazen, S.L. 1999. Eosinophil peroxidase nitrates protein tyrosyl residues.Implications for oxidative damage by nitrating intermediates ineosinophilic inflammatory disorders. Journal of Biological Chemistry.274:25933-25944). Protein content was determined by theMarkwell-modified Lowry protein assay (Markwell, M. A., Haas, S. M.,Bieber, L. L., and Tolbert, N. E. 1978. A modification of the Lowryprocedure to simplify protein determination in membrane and lipoproteinsamples. Analytical Biochemistry. 87:206-210) with bovine serum albuminas standard. The concentration of reagent H₂O₂ was determinedspectrophotometrically (ε₂₄₀=39.4 M⁻¹cm⁻¹; ref. (Nelson, D. P., andKiesow, L. A. 1972. Enthalpy of decomposition of hydrogen peroxide bycatalase at 25 degrees C. (with molar extinction coefficients of H₂O₂solutions in the UV). Analytical Biochemistry. 49:474-478).Myeloperoxidase (donor: hydrogen peroxide, oxidoreductase, EC 1.11.1.7)was initially purified from detergent extracts of human leukocytes bysequential lectin affinity and gel filtration chromatography asdescribed by Rakita (Rakita, R. M., Michel, B. R., and Rosen, H. 1990.Differential inactivation of Escherichia coli membrane dehydrogenases bya myeloperoxidase-mediated antimicrobial system. Biochemistry.29:1075-1080), and then trace levels of contaminating eosinophilperoxidase were then removed by passage over a sulphopropyl Sephadexcolumn (Wever, R., Plat, H., and Hamers, M. N. 1981. Human eosinophilperoxidase: a novel isolation procedure, spectral properties andchlorinating activity. FEBS Letters. 123:327-331). Purity of isolatedMPO was established by demonstrating a RZ of >0.84 (A₄₃₀/A₂₈₀), SDS PAGEanalysis with Coomassie Blue staining, and in-gel tetramethylbenzidineperoxidase staining (van Dalen, C. J., Whitehouse, M. W., Winterbourn,C. C., and Kettle, A. J. 1997. Thiocyanate and chloride as competingsubstrates for myeloperoxidase. Biochemical Journal. 327:487-492).Enzyme concentration was determined spectrophotometrically utilizing anextinction coefficient of 89,000 M⁻¹cm⁻¹/heme of MPO (Agner, K. 1972.Structure and function of oxidation-reduction enzymes. Tarrytown, N.Y.:Pergamon Press. 329-335). Delipidated and purified apoA-I was purchasedfrom Biodesign International (Saco, Me.) and used without furtherpurification following demonstration of purity by SDS-PAGE and silverstain analysis, and lack of significant free fatty acids by HPLC withon-line tandem mass spectrometry analysis (Zhang, R., Brennan, M. L.,Shen, Z., MacPherson, J. C., Schmitt, D., Molenda, C. E., and Hazen, S.L. 2002. Myeloperoxidase functions as a major enzymatic catalyst forinitiation of lipid peroxidation at sites of inflammation. Journal ofBiological Chemistry. 277:46116-46122). Low density lipoprotein (LDL;1.019<d<1.063 g/ml fraction) and high density lipoprotein (HDL;1.063<d<1.21 g/ml fraction) were isolated from fresh plasma bysequential ultracentrifugation (Hatch, F. T. 1968. Practical methods forplasma lipoprotein analysis. Advances in Lipid Research 6:1-68). Finalpreparations were extensively dialyzed against 50 mM sodium phosphate(pH 7.0), 200 μM diethylenetriaminepentaacetic acid (DTPA) and storedunder N₂ until use. LDL was acetylated with acetic acid anhydride(Goldstein, J. L., Ho, Y. K., Basu, S. K., and Brown, M. S. 1979.Binding site on macrophages that mediates uptake and degradation ofacetylated low density lipoprotein, producing massive cholesteroldeposition. Proceedings of the National Academy of Sciences of theUnited States of America. 76:333-337). NO₂Tyr immunostaining wasperformed as described (MacPherson, J. C., Comhair, S. A., Erzurum, S.C., Klein, D. F., Lipscomb, M. F., Kavuru, M. S., Samoszuk, M. K., andHazen, S. L. 2001. Eosinophils are a major source of nitricoxide-derived oxidants in severe asthma: characterization of pathwaysavailable to eosinophils for generating reactive nitrogen species.Journal of Immunology. 166:5763-5772). Specificity of immunostaining forNO₂Tyr was confirmed by showing loss of staining (i) in competitionstudies with 10 mM nitrotyrosine present during antibody—antigenincubations; and (ii) upon reduction of sample with dithionite.

Clinical specimens—Serum. For studies involving massspectrometry-dependent quantification of serum total protein, apoA-I andapoB-100 contents of NO₂Tyr and ClTyr, sequential patients (n=45) withcardiovascular disease (CVD) receiving care from the PreventiveCardiology Clinic of the Cleveland Clinic Foundation and healthyvolunteers (n=45) responding to advertisements were enrolled. CVD wasdefined clinically as coronary artery disease, peripheral arterialdisease, or cerebral vascular disease. Subjects with CVD were stable andwithout cardiac symptoms. Patients who experienced a myocardialinfarction or stroke within one month preceding enrolment wereineligible. Studies correlating HDL levels of NO₂Tyr and ClTyr withABCA1-dependent cholesterol efflux activities were performed on aseparate sequential set of subjects (n=12) without known (CVD) receivingcare from the Preventive Cardiology Clinic. All participants gavewritten informed consent and the Institutional Review Board of theCleveland Clinic Foundation approved the study protocol. Clinicalinvestigations were conducted in accordance with the Declaration ofHelsinki principles. A medical history and record review was performedto define coronary risk factors, including diabetes mellitus (defined byfasting blood glucose >125 mg/dl or hypoglycemic medications),hypertension (blood pressure >140/90 or anti-hypertensive medications inthe absence of known cardiac disease), family history of prematurecoronary heart disease (first degree relative with coronary heartdisease prior to age 60 by subject report), history ofhypercholesterolemia (fasting LDL cholesterol >160 mg/dl or lipidlowering medications in the absence of known cardiac disease), andcigarette smoking (any smoking within 1 year of study). A fasting bloodsample was obtained using a serum separator tube. Serum was isolated,aliquots placed into cryovials supplemented with antioxidant cocktailcomprised of butylated hydroxytoluene (100 μM final) and DTPA (2 mMfinal, pH 7.0), covered in argon and snap frozen at −80° C. until timeof analysis.

Immuno-affinity purification of nitrated proteins. An ImmunoPure ProteinA Orientation Kit (Pierce) was utilized to affinity-purify nitratedproteins from albumin/IgG depleted serum. Briefly, human serum (45 μl)was depleted of albumin and IgG using the ProteoPrep Albumin DepletionKit (Sigma-Aldrich Corp., St. Louis, Mo.) as recommended by themanufacturer. Affinity purified anti-nitrotyrosine antibody raisedagainst a synthetic octapeptide (Cys-Gly-NO₂Tyr-Gly-Gly-Gly-NO₂Tyr-Gly;SEQ ID NO: 11) was bound to protein A and cross-linked withdimethylpimelimidate. The albumin/IgG depleted patient serum was dilutedin 0.15 M NaCl, 0.1 M phosphate, pH 7.2, and applied to the column.Unbound protein fractions were eluted with 20 ml of PBS, and 10 ml of0.5 M NaCl. The bound proteins were eluted with 5 mM 3-nitrotyrosine in0.5 M NaCl. The bound fractions were concentrated using Centriprepfilter devices (YM-10, Millipore), dialyzed against 0.1 M urea, anddried down to a small volume using a Savant Instrument SpeedVacConcentrator (Savant Instruments Inc., Holbrook, New York). Protein inthe fractions was monitored with the bicinchoninic acid assay (Pierce),using BSA as standard.

2D SDS-PAGE. After dialysis, approximately 50 μg of protein was added to155 μl sample rehydration buffer and absorbed overnight onto 7 cm pH3-10 non-linear IPG ZOOM strips (Invitrogen). Isoelectric focusing wascarried out using the ZOOM IPG runner system from Invitrogen and theBiorad 3000V power supply using the following voltage step protocol: 100V for 30 min, 200 V for 20 min, 450 V for 15 min, 750 V for 15 min, and2000 V for 30 min. For the second dimension, focused IPG strips wereequilibrated in LDS sample buffer (Invitrogen) in the presence of NUPAGEsample reducing agent (Invitrogen) for 15 min, and an additionalincubation in LDS sample buffer in the presence of 125 mM iodoacetamidefor 15 min. The strips were placed on 4-12% bis-tris gels and embeddedin 0.5% agarose (w/v). The gels were stained for protein using eithercolloidal blue or silver staining. For immunoblotting gels weretransferred to 0.2 μm Immun-Blot PVDF membranes (Bio-Rad, Hercules,Calif.).

Clinical specimens—Tissue. LDL-like and HDL-like particles were isolatedfrom atherosclerotic lesions from aortas and femoral artery tissuesobtained at autopsy (tissue harvest within 10 h of death). Controlstudies to confirm that post mortem artifacts were not significantutilized vascular tissues (n=5) obtained fresh at the time of vascularsurgery. Normal human aortic tissues were obtained from transplantdonors. All tissues were immediately rinsed in ice-cold phosphatebuffered saline supplemented with 100 μM DTPA and immediately frozen inBuffer A (65 mM sodium phosphate, pH 7.4, 100 μM DTPA, 100 μM butylatedhydroxy toluene), under N₂ at −80° C. until analysis.

LDL- and HDL-like particle isolation and characterization from normalhuman aortic tissues and human atherosclerotic lesions. LDL- andHDL-like particles were isolated from fatty streaks and intermediatelesions of human thoracic aortae by sequential densityultracentrifugation (d=1.019-1.070 g/ml fraction for “lesion LDL,”1.063-1.21 g/ml fraction for “lesion HDL”) using a modification of themethod of Steinbrecher and Lougheed (Steinbrecher, U. P., and Lougheed,M. 1992. Scavenger receptor-independent stimulation of cholesterolesterification in macrophages by low density lipoprotein extracted fromhuman aortic intima. Arteriosclerosis & Thrombosis. 12:608-625) asdescribed (Krul, E. S., Tang, J., Kettler, T. S., Clouse, R. E., andSchonfeld, G. 1992. Lengths of truncated forms of apolipoprotein B(apoB) determine their intestinal production. Biochemical & BiophysicalResearch Communications. 189:1069-1076). “Control Aortic LDL” and“Control Aortic HDL”-like particles were similarly isolated fromresidual aortic tissues free of visible atherosclerotic plaque fromtransplant donors. A metal chelator (100 μM DTPA), myeloperoxidaseinhibitor (10 mM 3-aminotriazole), and protease cocktail comprised ofPMSF and Sigma protease inhibitor cocktail (catalog No P8340) wereincluded in all solutions used for lipoprotein isolation. Control Aorticand Lesion LDL and HDL were subjected to SDS-PAGE (Laemmli, U. K. 1970.Cleavage of structural proteins during the assembly of the head ofbacteriophage T4. Nature. 227:680-685) with Western blot analysis usingeither a rabbit anti-human apoB-100 antiserum (Hazen, S. L., andHeinecke, J. W. 1997. 3-Chlorotyrosine, a specific marker ofmyeloperoxidase-catalyzed oxidation, is markedly elevated in low densitylipoprotein isolated from human atherosclerotic intima. Journal ofClinical Investigation. 99:2075-2081), or goat anti-human apoA-I(Biodesign, Saco, Me.), respectively. Analysis of Control Aortic andLesion LDL-like particles with polyclonal antibody to apoB-100 detecteda 500 kDa protein, the mass of intact apolipoprotein B100. As previouslynoted (Krul, E. S., Tang, J., Kettler, T. S., Clouse, R. E., andSchonfeld, G. 1992. Lengths of truncated forms of apolipoprotein B(apoB) determine their intestinal production. Biochemical & BiophysicalResearch Communications. 189:1069-1076), both aggregated/cross-linkedand lower molecular mass forms of immunoreactive protein were alsopresent in LDL-like particles isolated from vascular tissues. SimilarWestern analyses were performed on Control Aortic and Lesion HDL-likeparticles using antibodies to apoA-I, confirming the presence of apoA-I.Analysis of Control Aortic and Lesion LDL-like particles by highresolution size exclusion chromatography (tandem Superose 6 and 12columns; Pharmacia LKB) demonstrated that immunoreactive apoB-100, totalcholesterol, and the majority of protein mass exhibited an apparentM_(r) similar to that of LDL isolated from plasma. Identity of apoA-I asa major protein constituent present in Control Aortic and LesionHDL-like particle preparations was also achieved by tandem MS sequenceanalysis following excision from Coomassie blue stained SDS PAGE gels.

Several control experiments indicated that post-mortem changes wereunlikely to contribute to apoA-I nitration and chlorination. First,control studies were performed on fresh arterial tissues harvested attime of vascular surgery (for lesion) and organ harvest fortransplantation (for normal/non-lesion arterial tissues). Comparablelevels of NO₂Tyr and ClTyr were noted within these freshly harvestedvascular tissues, compared to those obtained at autopsy. Second,following generation of powdered aortic tissues using a stainless steelmortar and pestle at liquid nitrogen temperatures, incubation of aortictissue powder (suspended in PBS) with MPO (100 nM) for 10 h at roomtemperature failed to increase levels of NO₂Tyr or ClTyr, as monitoredby both mass spectrometry and SDS-PAGE and Western analyses (forNO₂Tyr). Third, control studies demonstrated no significant formation of3-[¹³C₆]ClTyr or 3-[¹³C₆]NO₂Tyr in the above aortic tissue powder/MPOmixtures supplemented with L-[¹³C₆]tyrosine, incubated at roomtemperature for 10 h, and then subjected to mass spectrometry analysis.

Protein identification by mass spectrometry. Protein identificationswere carried out as previously described (Kinter, M., and Sherman, N.2000. Protein Sequencing and Identification Using Tandem MassSpectrometry, Willard, B. B., Ruse, C. I., Keightley, J. A., Bond, M.,and Kinter, M. 2003. Site-specific quantitation of protein nitrationusing liquid chromatography/tandem mass spectrometry. AnalyticalChemistry. 75:2370-2376). Briefly, bands were cut from Coomassie bluestained SDS-PAGE gels, reduced with DTT and alkylated withiodoacetamide. Protein was then digested in-gel by adding trypsin,peptides extracted, and then analyzed by capillary column HPLC-tandemmass spectrometry on an LCQDeca ion trap mass spectrometer system(ThermoFinnigan, San Jose, Calif.) equipped with a nanospray ionizationsource at a flow rate of 200 mL/min. Digest peptides were separated byreversed-phase capillary HPLC using a 50-μm-i.d. column with a 10-μm tippurchased from New Objective Corp. (Woburn, Mass.). The column waspacked with ˜6 cm of C18 packing material (Phenomenex, Torrence, Calif.)and eluted using a 45-min gradient of increasing acetonitrile (2-70%) in50 mM acetic acid. Protein identification was performed using adata-dependent analysis that acquired both mass spectra and CID spectrain a single run (Kinter, M., and Sherman, N. 2000. Protein Sequencingand Identification Using Tandem Mass Spectrometry, Willard, B. B., Ruse,C. I., Keightley, J. A., Bond, M., and Kinter, M. 2003. Site-specificquantitation of protein nitration using liquid chromatography/tandemmass spectrometry. Analytical Chemistry. 75:2370-2376). The searchprograms Sequest and Mascot were used for protein identifications.Manual sequence analyses were performed on select deuterium-enrichedpeptides during hydrogen-deuterium exchange mass spectrometry.

Nitrotyrosine and chlorotyrosine analyses—Protein-bound nitrotyrosineand chlorotyrosine were quantified by stable isotope dilution liquidchromatography—tandem mass spectrometry (Brennan, M. L., Wu, W., Fu, X.,Shen, Z., Song, W., Frost, H., Vadseth, C., Narine, L., Lenkiewicz, E.,Borchers, M. T., et al. 2002. A tale of two controversies: defining boththe role of peroxidases in nitrotyrosine formation in vivo usingeosinophil peroxidase and myeloperoxidase-deficient mice, and the natureof peroxidase-generated reactive nitrogen species. Journal of BiologicalChemistry. 277:17415-17427) on a triple quadrupole mass spectrometer(API 4000, Applied Biosystems, Foster City, Calif.) interfaced to aCohesive Technologies Aria LX Series HPLC multiplexing system (Franklin,Mass.). Synthetic [¹³C₆]-labeled standards were added to samples (eitheraliquots of serum, tissue/lesion homogenates, or bands visualized onPVDF membranes by colloidal blue stain and then excised) and used asinternal standards for quantification of natural abundance analytes.Simultaneously, a universal labeled precursor amino acid, [¹³C₉,¹⁵N₁]tyrosine, was added. Proteins were hydrolyzed under argonatmosphere in methane sulfonic acid, and then samples passed over minisolid-phase C18 extraction columns (Supelclean LC-C18-SPE minicolumn; 3ml; Supelco, Inc., Bellefone, Pa.) prior to mass spectrometry analysis.Results are normalized to the content of the precursor amino acidtyrosine, which was monitored within the same injection.Intrapreparative formation of both nitro[¹³C₉, ¹⁵N]tyrosine andchloro[¹³C₉, ¹⁵N]tyrosine was routinely monitored and negligible (i.e.<5% of the level of the natural abundance product observed) under theconditions employed.

Statistical Analysis. Power calculations were performed based onpreviously reported means and standard deviations of NO₂Tyr and ClTyr inclinical studies. It was determined that at least 30 patients wereneeded in each group to have 80% power to detect a 40%.

Example 1 Identification of Apolipoprotein A-I as a Nitrated Protein inSerum

Serum levels of protein-bound nitrotyrosine serve as a predictor ofatherosclerotic risk and burden in subjects (Shishehbor, M. H., Aviles,R. J., Brennan, M. L., Fu, X., Goormastic, M., Pearce, G. L., Gokce, N.,Keaney, J. F., Jr., Penn, M. S., Sprecher, D. L., et al. 2003.Association of nitrotyrosine levels with cardiovascular disease andmodulation by statin therapy. JAMA. 289:1675-1680), raising the questionof whether nitration of specific proteins might contribute to theatherosclerotic process. As a first step in investigating this question,we sought to determine the identities of nitrated proteins in serum.Samples from patients with CVD and controls were analyzed by SDS-PAGEand visualized by both Western blot analysis using anti-nitrotyrosineantibodies, and Coomassie blue staining for proteins. Comparison of thepattern of immunoreactivity seen in a Western blot vs. protein stainingrevealed that not all serum proteins are equally nitrated. An example ofa disparity between a modest abundance vs. an extensive degree ofnitration was reproducibly observed in a 29 kDa protein. This proteinband was cut from the Coomassie blue-stained gel, digested with trypsin,and unequivocally identified as apoA-I (NCBI accession number 253362)based upon the detection and sequencing of >30 peptides covering 96% ofthe protein sequence. Further confirmation of the identity of apoA-I asa nitrated protein was obtained through passage of serum through acolumn comprised of immobilized antibodies to nitrotyrosine, washing thecolumn with high salt, followed by elution with high salt supplementedwith 5 mM nitrotyrosine. Analysis of samples by 2-dimensional SDS-PAGEand capillary LC-tandem mass spectrometry-based sequencing confirmedapoA-I as a recovered protein (>90% coverage by LC/ESI/MS/MS). Furtherexamination of the anti-nitrotyrosine column eluent (high salt+5 mMnitrotyrosine) by 2-D SDS-PAGE followed by Western blot analysis usingantibodies to apoA-I provided additional complementary evidence ofapoA-I as a nitrated protein in vivo.

Example 2 Demonstration of Apolipoprotein A-I as a Preferred Target ofNitration and Chlorination within Serum, as Well as in Subjects withVersus without Cardiovascular Disease

Given the plethora of targets within tissues like serum and therelatively short diffusion distance for a reactive nitrogen species incomplex biological matrices, the apparent selective nitration of apoA-Iamongst serum proteins strongly suggested the existence of an enzymaticsource for NO-derived oxidants in close proximity to the lipoprotein invivo. One likely candidate was the enzyme MPO, since recent studies haveshown this enzyme both capable of catalyzing protein nitration in vivo(Brennan, M. L., Wu, W., Fu, X., Shen, Z., Song, W., Frost, H., Vadseth,C., Narine, L., Lenkiewicz, E., Borchers, M. T., et al. 2002. A tale oftwo controversies: defining both the role of peroxidases innitrotyrosine formation in vivo using eosinophil peroxidase andmyeloperoxidase-deficient mice, and the nature of peroxidase-generatedreactive nitrogen species. Journal of Biological Chemistry.277:17415-17427, Zhang, R., Brennan, M. L., Shen, Z., MacPherson, J. C.,Schmitt, D., Molenda, C. E., and Hazen, S. L. 2002. Myeloperoxidasefunctions as a major enzymatic catalyst for initiation of lipidperoxidation at sites of inflammation. Journal of Biological Chemistry.277:46116-46122, Baldus, S., Eiserich, J. P., Mani, A., Castro, L.,Figueroa, M., Chumley, P., Ma, W., Tousson, A., White, C. R., Bullard,D. C., et al. 2001. Endothelial transcytosis of myeloperoxidase confersspecificity to vascular ECM proteins as targets of tyrosine nitration.Journal of Clinical Investigation. 108:1759-1770, Gaut, J. P., Byun, J.,Tran, H. D., Lauber, W. M., Carroll, J. A., Hotchkiss, R. S., Belaaouaj,A., and Heinecke, J. W. 2002. Myeloperoxidase produces nitratingoxidants in vivo. Journal of Clinical Investigation. 109:1311-1319), aswell as playing a dominant role in generation of NO-derived oxidantsunder certain circumstances, such as within the extracellularcompartment at sites of inflammation (Brennan, M. L., Wu, W., Fu, X.,Shen, Z., Song, W., Frost, H., Vadseth, C., Narine, L., Lenkiewicz, E.,Borchers, M. T., et al. 2002. A tale of two controversies: defining boththe role of peroxidases in nitrotyrosine formation in vivo usingeosinophil peroxidase and myeloperoxidase-deficient mice, and the natureof peroxidase-generated reactive nitrogen species. Journal of BiologicalChemistry. 277:17415-17427). In order to both test the hypothesis thatMPO serves as a possible enzymatic catalyst for selective apoA-Initration in vivo, as well as quantitatively assess whether apoA-I isnitrated to a greater extent within subjects with CVD, sequentialpatients presenting to a cardiology clinic with documented CVD (n=45)and healthy control subjects (n=44) were consented and their serumsamples collected for analysis. The contents of both NO₂Tyr and ClTyrwere simultaneously quantified within total serum proteins, isolatedapoA-I and isolated apoB-100 utilizing stable isotope dilution HPLC withon-line electrospray ionization tandem mass spectrometry (LC/ESI/MS/MS).Table 2 lists the clinical and laboratory characteristics of thesubjects examined. As anticipated, patients with CVD were more likely tohave known CVD risk factors including history of diabetes, hypertension,smoking, family history of CVD, and history of hyperlipidemia. Subjectswith CVD also had lower LDL cholesterol levels and were more likely tobe on statin therapy, features likely attributable to ascertainment biasfrom enrollment of CVD subjects in a cardiology clinic.

TABLE 2 Clinical and laboratory characteristics Controls (n = 44) CVD (n= 45) p value Age 44.3 ± 11.1  65.6 ± 8.5 <0.001 Male gender (%) 24(54.6) 17 (37.8) 0.12 Diabetes (%) 0 (0)   24 (53.3) <0.001 Hypertension(%) 14 (31.8) 32 (71.1) <0.001 Smoking (%) 22 (50.0) 37 (82.2) 0.001Family hx CVD (%)  5 (11.4) 19 (42.2) 0.001 Hx hyperlipidemia (%)  7(15.9) 31 (68.9) <0.001 Statin use 0 (0)   28 (62.2) <0.001 TC (mg/dL)201 ± 33 165 ± 36 <0.001 HDLc (mg/dL)  60 ± 16  42 ± 15 <0.001 LDLc(mg/dL) 120 ± 34  91 ± 24 <0.001 TG (mg/dL) 108 ± 54 171 ± 96 <0.001Fasting Glucose (mg/dL)  93 ± 13 94 ± 3 0.43 Data are presented aseither percent or mean ± standard deviation as indicated. CVD =cardiovascular disease; HDLc = high density lipoprotein cholesterol; Hx= history of; LDLc = low density lipoprotein cholesterol; TC = totalcholesterol; TG = triglyceride.

Consistent with our recent published studies (Shishehbor, M. H., Aviles,R. J., Brennan, M. L., Fu, X., Goormastic, M., Pearce, G. L., Gokce, N.,Keaney, J. F., Jr., Penn, M. S., Sprecher, D. L., et al. 2003.Association of nitrotyrosine levels with cardiovascular disease andmodulation by statin therapy. JAMA. 289:1675-1680), serum NO₂Tyr contentwas significantly increased approximately 1.5-fold (p<0.001) in subjectswith CVD relative to that of healthy controls (Table 3). Similar results(2-fold increase; p=0.001 for CVD vs. controls, Table 3) were observedwhen analyzing the NO₂Tyr content of isolated apoB-100, the majorprotein constituent of LDL and a lipoprotein reported to bind to MPO invitro (Carr, A. C., Myzak, M. C., Stocker, R., McCall, M. R., and Frei,B. 2000. Myeloperoxidase binds to low-density lipoprotein: potentialimplications for atherosclerosis. FEBS Letters. 487:176-180, Yang, C.Y., Gu, Z. W., Yang, M., Lin, S. N., Garcia-Prats, A. J., Rogers, L. K.,Welty, S. E., and Smith, C. V. 1999. Selective modification of apoB-100in the oxidation of low density lipoproteins by myeloperoxidase invitro. Journal of Lipid Research. 40:686-698). Remarkably, a 70-foldenrichment in NO₂Tyr content was noted within serum apoA-I relative toserum total proteins and isolated apoB-100. Moreover, a significantincrease in NO₂Tyr content of apoA-I was also noted in serum from CVDvs. healthy control subjects (p=0.005; Table 2). Parallel analyses ofsamples for ClTyr content, a protein modification specific forMPO-catalyzed oxidation (Hazen, S. L., Hsu, F. F., Gaut, J. P., Crowley,J. R., and Heinecke, J. W. 1999. Modification of proteins and lipids bymyeloperoxidase. Methods in Enzymology 300:88-105, Brennan, M. L., Wu,W., Fu, X., Shen, Z., Song, W., Frost, H., Vadseth, C., Narine, L.,Lenkiewicz, E., Borchers, M. T., et al. 2002. A tale of twocontroversies: defining both the role of peroxidases in nitrotyrosineformation in vivo using eosinophil peroxidase andmyeloperoxidase-deficient mice, and the nature of peroxidase-generatedreactive nitrogen species. Journal of Biological Chemistry.277:17415-17427, Hazen, S. L., and Heinecke, J. W. 1997.3-Chlorotyrosine, a specific marker of myeloperoxidase-catalyzedoxidation, is markedly elevated in low density lipoprotein isolated fromhuman atherosclerotic intima. Journal of Clinical Investigation.99:2075-2081, Brennan, M. L., Anderson, M., Shih, D., Qu, X., Wang, X.,Mehta, A., Lim, L., Shi, W., Hazen, S. L., Jacob, J., Crowley, J.,Heinecke, J. W., and Lusis, A. J. 2001. Increased atherosclerosis inmyeloperoxidase-deficient mice. J. Clin. Invest. 107:419-30), revealedrelatively low levels within total proteins and isolated apoB-100 fromserum compared to over 100-fold enrichment in ClTyr content noted withinisolated apoA-I (Table 2). Further, while trends for increases in ClTyrcontent within subjects with CVD were noted in total protein andisolated apoB100, these differences failed to reach statisticalsignificance. In contrast, significant increases in ClTyr content wereobserved within apoA-I recovered from serum (p<0.001; Table 3).

TABLE 3 Apolipoprotein A-I is a preferred target for nitration andchlorination in serum and in cardiovascular disease NitrotyrosineChlorotyrosine median (IQR) median (IQR) (μmol oxTyr/ (μmol oxTyr/ molTyr) p vaue mol Tyr) p value Section 1.01 Serum total protein Control6.1 [3.9-7.8] 1.6 [0.6-2.4] CVD  9.0 [5.7-12.9] <0.001 1.9 [1.3-3.1]0.07 apoB-100 Control 4.0 [1.3-6.9] 0.0 [0.0-1.9] CVD  8.7 [5.2-12.1]0.001 1.9 [0.1-4.0] 0.24 Section 1.02 apoA-I Control 438 [335-598] 186[114-339] CVD 629 [431-876] 0.005 500 [335-765] <0.001Aliquots of serum (100 ug protein) from the entire cohort from Table 2(CVD and healthy control subjects) were either analyzed directly (fortotal protein) or resolved by SDS PAGE, transferred to PVDF membranes,and bands corresponding to apoA-I and apoB-100 visualized, excised, andthen analyzed by stable isotope dilution LC/ESI/MS/MS, as describedunder Methods. Results shown are for median and interquartile ranges ofnitrotyrosine and chlorotyrosine contents of total serum protein or theindicated lipoprotein, expressed as the mole ratio of oxidized aminoacid to parent amino acid, tyrosine. The p values shown are forcomparisons of NO₂Tyr and ClTyr content between Control and CVD groupswithin the corresponding indicated protein(s). apo=apolipoprotein;CVD=cardiovascular disease; IQR=interquartile range.

The strength of the relationship between the content of NO₂Tyr and ClTyrwithin total proteins and isolated apoA-I from serum was furtherexamined in the entire cohort (CVD plus controls). As shown in Table 4(top), increasing NO₂Tyr content was associated with increasingfrequency of CVD, as monitored in either total serum proteins orisolated apoA-I from serum. Further, comparisons between subjects withhigher vs. lower levels of NO₂Tyr (third vs. first tertile) demonstratedapproximately 6-fold increase in odds for having CVD, whether examiningtotal proteins or isolated apoA-I from serum (Table 3, bottom). Incontrast, only the ClTyr content of isolated apoA-I, and not ClTyrcontent of total serum proteins (or apoB-100, not shown), was associatedwith increasing frequency or odds of CVD within the cohort. Remarkably,subjects possessing a high (top tertile) apoA-I ClTyr content were16-fold more likely to have CVD than those with low (bottom tertile)apoA-I ClTyr content (Table 4, bottom).

TABLE 4 Relationship between serum total protein and apolipoprotein A-Initrotyrosine and chlorotyrosine content with cardiovascular diseaseprevalence Frequency of CVD per Tertile Serum 1 2 3 p for trend Totalprotein NO₂Tyr 30.8% 33.3% 69.2% 0.005 apoA-I NO₂Tyr 32.0% 44.0% 72.0%0.005 1.total protein ClTyr 40.0% 50.0% 58.3% 0.20 apoA-I ClTyr 20.0%48.0% 80.0% <0.001 Odds Ratio (95% CI) of CVD per Tertile Serum 1 2 3Total protein NO₂Tyr 1.0 1.1 (0.4-3.6) 5.1 (1.6-16.4) apoA-I NO₂Tyr 1.01.7 (0.5-5.3) 5.5 (1.6-18.4) 2.total protein ClTyr 1.0 1.5 (0.5-4.6) 2.1(0.7-6.6)  apoA-I ClTyr 1.0  3.7 (1.1-13.0) 16.0 (4.0-64.0)  Displayedare (top) frequencies of cardiovascular disease prevalence within eachtertile of the entire cohort; and (bottom) odds ratios and 95%confidence intervals for second and third tertiles compared to thelowest (first) tertile as predictors of CVD. apo = apolipoprotein; CI =confidence interval; ClTyr = chlorotyrosine; CVD = cardiovasculardisease; NO₂Tyr = nitrotyrosine.

Example 3 Demonstration of Apolipoprotein A-I as a Preferred Target ofNitration and Chlorination within Human Atherosclerotic Lesions

To examine whether the preferential targeting of apoA-I by NO⁻ andMPO-generated oxidants occurred within human atheroma, additionalstudies were performed examining total proteins, apoB-100 and apoA-Irecovered from human aortic tissues. LDL-like and HDL-like particleswere isolated from both normal aortic tissues and atherosclerotictissues by differential buoyant density centrifugation, and thenconfirmed to be enriched both in cholesterol and the appropriateapolipoprotein preparation by Western analyses using polyclonalantibodies to either apoB-100 or apoA-I, as described under “Methods”.The majority of apoA-I recovered within HDL-like particles fromatherosclerotic lesions was monomeric. More quantitative assessments ofNO₂Tyr and ClTyr contents of apoA-I recovered from normal aortic andlesion tissues relative to that observed in aortic and lesion totalproteins and apoB-100 were obtained by stable isotope dilutionLC/ESI/MS/MS analyses, the results for which are shown in Table 4. Ofnote, the contents of NO₂Tyr and ClTyr within total proteins, apoB-100and apoA-I recovered from normal aortic tissues and humanatherosclerotic lesions were higher than that observed in serum (compareTables 3 versus 5), suggesting protein modification by NO- andMPO-generated oxidants preferentially occurs within the artery wall,particularly within atherosclerotic lesions, compared to theintra-vascular (blood) compartment. As was observed within serum and theserum-derived isolated lipoproteins, the contents of both NO₂Tyr andClTyr in lesion apoA-I demonstrated a dramatic selective enrichmentrelative to lesion total proteins and lesion apoB-100 (Table 5).Similarly, higher levels are observed within total proteins and theisolated lipoproteins from diseased vs. normal vascular tissues.

TABLE 5 Apolipoprotein A-I is a preferred target for nitration andchlorination within human aortic atherosclerotic lesions NitrotyrosineChlorotyrosine Median (IQR) Median (IQR) (μmol oxTyr/mol Tyr) p value(μmol oxTyr/mol Tyr) p value Section 1.03 Normal total protein   55[24-143]   63 [25-128] apoB-100   97 [43-222] 0.57   49 [21-121] 0.93Section 1.04 apoA-I   401 [185-637] <0.001   678 [299-1,311] <0.001Section 1.05 Lesion total protein   108 [51-346]   232 [111-431]apoB-100   255 [91-480] 0.67   318 [59-385] 0.92 Section 1.06 apoA-I2,340 [1,665-5,050]* <0.001 3,930 [1,679-7,005]* <0.001

Specimens of normal human aorta (n=10 subjects) and human aorticatherosclerotic tissues (n=22 subjects) were stripped of adventia, andthen, pulverized into a powder in stainless steel mortar and pestle atliquid nitrogen temperatures, and the contents of nitrotyrosine andchlorotyrosine analyzed by stable isotope dilution LC/ESI/MS/MS, asdescribed under Methods. Total protein content of biomarkers wasascertained using powdered human vascular tissue. The contents ofoxidized amino acids within normal aortic and atheroscleroticlesion-derived apoB-100 and apoA-I were assessed following isolation ofLDL-like and HDL-like particles from powdered vascular tissues bysequential buoyant density centrifugation, further resolving by SDSPAGE, transfer to PVDF membranes, and then bands corresponding to apoA-Iand apoB-100 visualized, excised, and analyzed by stable isotopedilution LC/ESI/MS/MS, as described under Methods. Results shown are formedian and interquartile ranges of nitrotyrosine and chlorotyrosinecontents of normal aortic and lesion total protein or the indicatedlipoproteins, expressed as the mole ratio of oxidized to parent aminoacid, tyrosine. The p values shown are for comparisons of nitrotyrosineor chlorotyrosine content in the indicated isolated lipoproteins fromhuman normal aortic and atherosclerotic lesions vs. the correspondingcontent observed in normal or lesion aortic tissue total proteins.*P<0.001 for comparison of lesional apoA-I versus normal aortic tissueapoA-I.

The present studies provide both the first direct evidence of apoA-I,the major protein constituent of HDL, as a preferential target fornitration and chlorination in the artery wall, as well as a potentialmechanism(s) for generation of a pro-atherogenic form of HDL. Theremarkable selective enrichment in apoA-I NO₂Tyr and ClTyr contentobserved both within human atherosclerotic lesions and the systemiccirculation indicate that NO-derived oxidants and MPO-catalyzedreactions selectively target the lipoprotein for oxidative modification.We observed a combined ox-amino acid (ox-AA) content of 5.500 μmolox-AA/mol Tyr within lesional apoA-I (Table 5). Given there are 7tyrosine residues per apoA-I and up to 4 apoA-I molecules per HDLparticle, we calculate that on average, approximately 15% of theHDL-like particles recovered from human aortic lesions possess at leastone oxidative modification (i.e. 5.5 ox-AA/10³ Tyr×7 Tyr/apoA-I×4apoA-I/HDL particle=15.4%). If one looks a the top quartile values,which demonstrated a combined ox-AA content ranging between 10,000 to25.000 μmol ox-AA/mol Tyr, then a remarkable 28% to 50% of lesional HDLin this top quartile possess either a ClTyr or a NO₂Tyr residue. SinceMPO, HOCl-modified proteins, and apoA-I all co-localize within theprotected environment of the subendothelial compartment, it is not hardto imagine that this number may be even higher in some locations. Whilethe selective enrichment of MPO-generated oxidation products was mostpronounced within circulating HDL of individuals with CVD and humanatherosclerotic lesions, marked enrichment was also noted within apoA-Irecovered from both serum of healthy subjects and transplant donoraortic tissues, suggesting a potential physiologicanti-inflammatory/anti-oxidant role for apoA-I binding of MPO andscavenging of MPO-generated oxidants.

B. apoA-I Peptide Fragment Studies

The present studies establish the molecular events associated withMPO-mediated modification of apoA-I and specifically relate those eventsto alterations of apoA-I function. We have mapped the sites ofMPO-mediated nitration and chlorination with tandem mass spectrometryand now demonstrate the specific modification of two regions of theprotein. We also establish the hierarchy of modifications and report acolocalization of the preferred residue modified by both MPO-catalyzedchlorination and nitration with the MPO-interaction site in the helix 8region. A strong correlation is noted between dose-dependent progressionof MPO-mediated site-specific modifications of apoA-I and loss of bothABCA1-dependent reverse cholesterol transport and inhibition of apoA-Ilipid binding. Our data suggest a link between the degree ofMPO-catalyzed site-specific apoA-I modifications and the loss ofimportant anti-atherosclerotic functions of HDL. MPO-catalyzed oxidativemodification of apoA-I in the artery wall may thus contribute to theclinical association between MPO and cardiovascular disease.

Experimental Procedures

HDL and apoA-I Modification Reactions.

Whole blood was drawn from healthy donors who gave written informedconsent for a study protocol approved by the Institutional review boardof the Cleveland Clinic Foundation. HDL was isolated from human plasma(density range—1.063 g/mL to 1.210 g/mL) using differentialultra-centrifugation and extensively dialyzed in 50 mM phosphate buffer(pH 7.0) with 100 μM diethylenetriamine pentaacetic acid (DTPA) in thedark at 4° C. Delipidated and purified apoA-I was purchased fromBiodesign International (Saco, Me.) and used without furtherpurification.

The MPO-mediated modification reactions were carried out in a 50 mMphosphate buffer, pH 7.0, containing 100 μM DTPA, 1 mg/mL protein(apoA-I), 57 nM purified human MPO (donor: hydrogen peroxide,oxidoreductase, EC 1.11.1.7; A₄₃₀/A₂₈₀ ratio of 0.79), and either 1 mMnitrite (for the nitration reactions) or 100 mM chloride (for thechlorination reactions). The myeloperoxidase reactions were initiated byadding hydrogen peroxide at varying concentrations (0-200 μM) andcarried out at 37° C. for 1 h. These reaction conditions includephysiologically relevant amounts of MPO, chloride and nitrite, andhydrogen peroxide concentrations that range from physiologic topathologic.

The peroxynitrite and HOCl reactions were similarly carried out at 37°C. for 1 h in 50 mM phosphate buffer, pH 7.0, containing 100 μM DTPAwith the peroxynitrite and HOCl added to give final concentrationsbetween 0 and 200 μM.

In all reactions, the concentrations of the key reactants was verifiedspectrophotometrically using molar extinction coefficients of 170cm⁻¹mM⁻¹ at 430 nm for MPO, 39.4 cm⁻¹M⁻¹ at 240 nm for hydrogenperoxide, 350 cm⁻¹M⁻¹ at 292 nm for HOCl (NaOCl), and 1670 cm⁻¹M⁻¹ at302 nm for peroxynitrite.

The modified proteins were taken immediately for the apoA-I functionalstudies described below. An aliquot of each reaction was removed,precipitated with acetone, and separated by SDS-PAGE for the massspectrometry experiments.

Mass Spectrometry Experiments.

Protein digestion. The protein bands were digested according to anin-gel digestion procedure (Kinter, M., and Sherman, N.E. (2000) ProteinSequencing and Identification Using Tandem Mass Spectrometry. John Wileyand Sons, New York). Briefly, the protein bands were cut from the geland washed in 50% ethanol/5% acetic acid prior to tryptic digestion witha modified, sequencing grade trypsin (Promega, Madison, Wis.) overnightat room temperature. The peptides were extracted from the gel,evaporated to dryness, and reconstituted in either 1% acetic acid or0.1% formic acid for analysis by capillary column HPLC-electrosprayionization mass spectrometry.

Identification of the modification sites. The detailed mapping anddetection of the nitration and chlorination sites was carried out usingan LCQ Deca ion trap mass spectrometer system (ThermoFinnigan, San Jose,Calif.) equipped with a nanospray ionization source (Protana, Odense,Denmark). The source was operated under microspray conditions at a flowrate of 200 nl/min. The digests were analyzed by reversed-phasecapillary HPLC using a 50-μm i.d. column with a 15-μm i.d. tip purchasedfrom New Objective Corp. (Woburn, Mass.). The column was packed with ˜6cm of C18 packing material (Phenomenex, Torrence, Calif.) and elutedusing a 45-min gradient of increasing acetonitrile (2-70%) in 50 mMacetic acid. The data were acquired in the data-dependent mode,recording a mass spectrum and three collision-induced dissociation (CID)spectra in repetitive cycles (Kinter, M., and Sherman, N.E. (2000)Protein Sequencing and Identification Using Tandem Mass Spectrometry.John Wiley and Sons, New York). The program Sequest was used to compareall CID spectra recorded to the sequence of human apoA-1 (NCBI accessionnumber 229513) and considering the appropriate changes in the tyrosineresidues masses of +34 Da for chlorination and +45 Da for nitration.

Site-Specific Quantitation of Protein Modification.

Site-specific quantitation experiments were performed on a QToFmicromass spectrometry system (Waters, Milford, Mass.) equipped with a CapLCHPLC system (Waters) with autoinjector. The electrospray ionizationsource was operated under microspray conditions at a flow rate of 600mL/min. The digests were analyzed by reversed-phase capillary HPLC usinga 75-μm i.d. column with a 19-μm i.d. nanospray source capillary. Thecolumn was packed with ˜15 cm of 10-μm C18 packing material (Phenomenex,Torrence, Calif.) and eluted using a 45-min gradient of increasingacetonitrile (2-70%) in 0.1% formic acid. Quantitation was achieved byusing the Native Reference Peptide method developed for thesite-specific quantitation of post-translational modifications (38,39)in the selected ion monitoring mode (SIM). For the SIM experiments, fullscans from m/z 300 to m/z 1600 were acquired with the time-of-flightmass analyzer of the QToF instrument and mass chromatograms constructedbased the m/z value of the different peptide ions of interest. TheapoA-I peptides ATEHLSTLSEK, SEQ ID NO: 9, and QGLLPVLESFK, SEQ ID NO:10 were used as native reference peptides. The relative quantity of theeach analyte peptide was determined by dividing the chromatographic peakarea of that analyte peptide by the chromatographic peak area of thereference peptide.

ApoA-I Functional Studies.

Cholesterol efflux. The cholesterol efflux experiments were performedaccording to established procedures (Smith, J. D., Miyata, M., Ginsberg,M., Grigaux, C., Shmookler, E., and Plump, A. S. (1996) J. Biol. Chem.271, 30647-30655, Takahashi, Y., and Smith, J. D. (1999) Proc. Natl.Acad. Sci. U.S.A. 96, 11358-11363). Subconfluent RAW264.7 cells in24-well dishes were cholesterol loaded and labeled overnight in 0.5 mLof DGGB (DMEM supplemented with 50 mM glucose, 2 mM glutamine, and 0.2%BSA), containing [³H]-cholesterol-labeled acetylated low densitylipoprotein (AcLDL). The [³H]-cholesterol-labeled AcLDL was prepared byincubating [³H]-cholesterol for 30 min at 37° C. with the AcLDL anddiluted in DGGB to give a final concentration of 50 μg/ml AcLDL with0.33 μCi/ml [³H]-cholesterol. The day after labeling, the cells werewashed three times in PBS, 0.2% BSA and incubated with 0.5 mL of DGGBwith or without 0.1 mM 8-Br-cAMP for 16 h. After the 16-h incubation, 50μg/mL of HDL protein in 0.5 mL DGGB with or without 8-Br-cAMP was addedto each well. After a 4-h incubation at 37° C., 100 μL of medium wasremoved, centrifuged, and the radioactivity counted as a measure of theeffluxed cholesterol in the media. The respective cells from each wellwere extracted with hexane/isopropanol (3:2, v:v) and the radioactivitydetermined as a measure of the cholesterol retained in the cell. Thepercentage of cholesterol effluxed was calculated as the radioactivityin the medium divided by the total radioactivity (medium radioactivityplus cell radioactivity).

Lipid binding. An LDL aggregation assay was used to test apoA-I lipidbinding, modified from a previously described assay (Liu, H., Scraba, D.G., and Ryan, R. O. (1993) FEBS Lett. 316, 27-33). In a 96-well assayplate, 75 μg of LDL was mixed with or without 3 μg of control ormodified apoA-I in a final volume of 200 μl of reaction buffer (50 mMTris-HCl, pH 7.4, with 150 mM NaCl and 2 mM CaCl₂). Each reaction wasperformed in triplicate. The plate was then incubated in a SpectraMaxplate reader (Molecular Devices, Sunnyvale, Calif.) at 37° C. for 10min. A 20 μL aliquot of diluted phospholipase C, derived from Bacilluscereus (Sigma P7147), sufficient to induce aggregation in 1 hr, or abuffer control, was added to each well to hydrolyze the phospholipidpolar head groups to make the LDL surface hydrophobic and initiateaggregation. Aggregation at 37° C. was monitored by absorbance at 478nm, read every 2 minutes for a period of 1 hr. ApoA-I lipid bindingactivity results in the inhibition of the LDL aggregation, and wascalculated from the aggregation rate (ΔO.D./min.) during the rapid phasethat occurred after a short time lag. The aggregation rate for themodified apoA-I was normalized to the rate of native apoA-I.

Detection of Modified apoA-I in Human Atheroma.

ApoA-I isolation. Human atheroma tissue was isolated from aortasobtained at autopsy within 10 h of death. The tissue was immediatelyrinsed in ice-cold phosphate buffered saline supplemented with 100 μMDTPA and immediately frozen in 65 mM sodium phosphate, pH 7.4, with 100μM DTPA and 100 μM butylated hydroxy toluene, under N₂ at −80° C. untilanalysis.

Fatty streaks and intermediate lesions of human thoracic aorta werepowered using a stainless steel mortar and pestle at liquid nitrogentemperatures and mixed with PBS, containing 100 μM DTPA and a proteaseinhibitor cocktail (Sigma catalog No P8340) for 10 hours at 4° C. Thesuspended lesion sample was centrifuged and the supernatant used forapoA-I purification. For the purification, apoA-I was bound to ananti-HDL IgY resin (GenWay Biotech, San Diego, Calif.), eluted in 0.1Mglycine (pH 2.5), and the eluate neutralized by addition of 1M tris (pH8.0). The neutralized sample was dissolved in sample loading bufferwithout heating, run in a 12.5% SDS-PAGE gel (Criterion, BioRadLaboratories), and detected by Coomassie blue-staining. The SDS-PAGEanalysis revealed that >90% of the protein recovered from the column wasapoA-I.

LC-tandem MS analysis. The immunoaffinity isolated apoA-I band was cutfrom the gel and digested with trypsin as described above. The LC-tandemMS experiments used a ThermoFinnigan LTQ linear ion trap massspectrometer with a Surveyor HPLC pump and autosampler system. Sampleswere injected onto a 10-cm×75 μm i.d. capillary column that was elutedwith a linear gradient of acetonitrile in 50 mM acetic acid atapproximately 1 μL/min. Selected reaction monitoring experiments (SRM)were used to record the product ion spectra of molecular ion of peptidescontaining each modified tyrosine residue characterized in the mappingexperiments. Chromatograms for those peptides were reconstructed byplotting the fragmentation of the molecular ions to the most abundantproduct ions in the respective CID spectra. Detection of the appropriatepeptide was verified by the CID spectrum that was recorded at thatretention time.

Statistical Analyses.

Statistically significant differences were determined by either aone-way analysis of variance using a Tukey-Kramer multiple comparisonstest or a Student's t-test. Statistical significant differences arereported when p<0.05.

Example 4 Mapping the Nitration and Chlorination Sites in apoA-I

Initial experiments focused on determining the tyrosine nitration sitesin apoA-I produced by treating HDL with both an enzymatic modificationsystem (MPO/H₂O₂/NO₂ ⁻) with varying concentrations of hydrogenperoxide, and a non-enzymatic system using varying concentrations ofperoxynitrite. After each treatment, the proteins in HDL wereprecipitated with cold acetone, separated by SDS-PAGE, and the apoA-Iband cut from the gel for in-gel digestion with trypsin. The digest wasanalyzed by capillary column HPLC-tandem mass spectrometry using thedata-dependent mode of the ion trap mass spectrometry system.Approximately 2000 CID spectra were recorded and these spectra weresearched for the spectra of modified peptides. The search routine wasfocused on the amino acid sequence of apoA-I and used a tyrosine residuemass difference of +45 Da to find the spectra of the nitrated peptides.These analyses detected peptides that covered 95% of the proteinsequence, including peptides that contained all 7 of the tyrosineresidues in the mature apoA-I sequence. Two peptides containingnitrotyrosine residues (Y192 and Y166) were found in HDL treated withthe MPO/H₂O₂/NO₂ ⁻ at concentrations of hydrogen peroxide <50 μM. Athigher hydrogen peroxide concentrations (>100 μM), additionalnitrotyrosine-containing peptides (Y29 and Y236) could also be detected.The three remaining tyrosine residues are contained in peptides thatwere detected and sequenced in these analyses, but no correspondingnitrated form was found under any reaction condition that was tested. Asa comparison, a similar reaction of HDL with 100 peroxynitrite gavenitrotyrosine modifications of the apoA-I at three tyrosine residues;Y166, Y18, and Y236. Again, the remaining tyrosine residues weredetected in these analyses exclusively in the un-nitrated form.

The CID spectra of the five nitrated peptides are shown in FIG. 1. Foreach peptide, the identity is established by the series of product ionsrecorded in the respective CID spectrum. A component of these CIDspectra is the characteristic residue mass of the nitrotyrosine moiety(208 Da). Overall, the combination of the peptide molecular weightmeasurements, the peptide sequence information in the CID spectra, andthe known apoA-I amino acid sequence allows unambiguous assignment ofthe nitrotyrosine positions.

Subsequent experiments used complementary protein chlorination systems,with both enzymatic (MPO/H₂O₂/Cl⁻) and non-enzymatic (HOCl) reactions,to modify the HDL. Four sites of chlorination were detected in both ofthese reactions, Y192, Y166, Y29, and Y236. These are the same tyrosineresidues that were nitrated by the MPO/H₂O₂/NO₂ ⁻ reaction. As noted forthe nitration sites, chlorination of Y192 and Y166 was detected athydrogen peroxide concentrations <50 μM whereas chlorination of Y29 andY236 required >100 μM hydrogen peroxide. The Y18 nitration site seenwith peroxynitrite treatment was not chlorinated by either the MPO- orHOCl-mediated reaction. FIG. 2 contains the CID spectra of the fourchlorinated peptides. These CID spectra are characterized by afragmentation pattern that includes the residue mass of thechlorotyrosine (197 Da for the more abundant ³⁵Cl isotope). As noteabove for the nitrated peptides, the combination of the peptidemolecular weight, the information in CID spectrum, and the apoA-I aminoacid sequence gives a clear-cut assignment of the chlorotyrosinepositions.

Example 5 Quantitative Analyses Identify the Preferred ModificationSites

A comprehensive map of the nitration and chlorination sites in apoA-I isshown in FIG. 3. One observation made during the initial mappingexperiments was a potential hierarchy in the various modifications, withtwo of the MPO-mediated nitration/chlorination sites being modifiedprior to the other two, one MPO-mediated modification site that was notmodified by peroxynitrite, and one peroxynitrite modification site thatwas not modified by MPO. As a result, quantitative experiments weredesigned to specifically determine the order of the nitration andchlorination sites.

These quantitative experiments used our previously described NativePeptide Reference method to follow the disappearance of the differentregions of apoA-I, represented as the respective tyrosine-containingpeptides formed by the trypsin digestion (Willard, B. B., Ruse, C. I.,Keightley, J. A., Bond, M., and Kinter, M. (2003) Anal. Chem. 75,2370-2376, Ruse, C. I., Willard, B., Jin, J. P., Haas, T., Kinter, M.,and Bond M. (2002) Anal. Chem. 74, 1658-1664). HDL was treated undernitration or chlorination conditions with increasing amounts of H₂O₂ inthe MPO-mediated reactions, or increasing amounts of peroxynitrite andHOCl in the non-enzymatic reactions. The reactions were stopped byprotein precipitation with −20° C. acetone and separated by SDS-PAGE.The apoA-I bands were cut for tryptic digestion and the digests analyzedby capillary column LC-ESI-MS. The progression of MPO-mediatedmodification of the individual sites in apoA-I was monitored by plottingmass chromatograms for each tyrosine containing peptide and calculatingthe respective peak area ratios relative to the unmodified nativereference peptide. As shown in FIGS. 4B and 5B, progression ofMPO-mediated modification, with increasing concentrations of H₂O₂, ateach site in apoA-I produces a decreased recovery of the respectiveunmodified peptides in the digest. These data revealed that Y192 servesas the preferred MPO-catalyzed nitration and chlorination site, followedby Y166 and Y29, and with only a minor degree of modification of Y236 athigher levels of oxidant. A comparable pattern of oxidative modificationwas also seen in the HOCl reaction. Remarkably, the relativeeffectiveness of the HOCl reaction was significantly less than that ofthe MPO-catalyzed chlorination reaction. Specifically, the MPO-catalyzedoxidation reaction produced greater modification at every concentrationof H₂O₂ examined relative to molar equivalent amounts of HOCl. Incontrast, the dose response characteristics of the peroxynitritereaction differed both in the efficiency of the modification reactionand in the clear preference for nitration of Y18 and the absence ofnitration at Y192.

Example 6 Oxidatively Modified apoA-I is Functionally Impaired

FIGS. 4A and 5A also show the dose-response effects of modification ofHDL on ABCA1-mediated cholesterol efflux properties. HDL was treatedwith the MPO/H₂O₂/NO₂ ⁻ system, peroxynitrite, the MPO/H₂O₂/Cl⁻ system,or HOCl. Cholesterol efflux was then measured by incubation of thetreated samples with cholesterol loaded murine macrophage RAW264.7 cellsin the presence and absence of pretreatment with 8Br-cAMP. In theabsence of 8Br-cAMP treatment, RAW264.7 cells do not express anappreciable level of ABCA1 and support ABCA1-independent cholesterolefflux to HDL but no cholesterol efflux to apoA-I (Smith, J. D., Miyata,M., Ginsberg, M., Grigaux, C., Shmookler, E., and Plump, A. S. (1996) J.Biol. Chem. 271, 30647-30655, Remaley, A. T., Stonik, J. A., Demosky, S.J., Neufeld, E. B., Bocharov, A. V., Vishnyakova, T. G., Eggerman, T.L., Patterson, A. P., Duverger, N. J., Santamarina-Fojo, S., and Brewer,H. B., Jr. (2001) Biochem. Biophys. Res. Comm. 280, 818-823, Takahashi,Y., Miyata, M., Zheng, P., Imazato, T., Horwitz, A., and Smith, J. D.(2000) Biochim. Biophys. Acta. 1492, 385-394, Chen, W., Sun, Y., Welch,C., Gorelik, A., Leventhal, A. R., Tabas, I., and Tall, A. R. (2001) J.Biol. Chem. 276, 43564-43569). 8Br-cAMP treatment of RAW264.7 cellsinduces ABCA1 mRNA and protein allowing ˜2-fold higher cholesterolefflux to HDL and significant levels of cholesterol efflux to lipid-freeapoA-I (Smith, J. D., Miyata, M., Ginsberg, M., Grigaux, C., Shmookler,E., and Plump, A. S. (1996) J. Biol. Chem. 271, 30647-30655, Takahashi,Y., and Smith, J. D. (1999) Proc. Natl. Acad. Sci. U.S.A. 96,11358-11363, Remaley, A. T., Stonik, J. A., Demosky, S. J., Neufeld, E.B., Bocharov, A. V., Vishnyakova, T. G., Eggerman, T. L., Patterson, A.P., Duverger, N. J., Santamarina-Fojo, S., and Brewer, H. B., Jr. (2001)Biochem. Biophys. Res. Comm. 280, 818-823, Takahashi, Y., Miyata, M.,Zheng, P., Imazato, T., Horwitz, A., and Smith, J. D. (2000) Biochim.Biophys. Acta. 1492, 385-394). Therefore, the presence or absence of8Br-cAMP pretreatment allows one to measure both ABCA1-dependent and-independent cholesterol efflux. As seen in FIGS. 7A and 8A, theMPO-mediated nitration and chlorination reactions, and the HOCltreatment, produced dose-dependent losses of the ABCA1-dependentcholesterol efflux to HDL without affecting ABCA1-independent efflux toHDL. Overall, the rank order of efficiency for functional impairment byvarious modification reactions was MPO-mediated chlorination >HOClchlorination >MPO-mediated nitration >>peroxynitrite nitration.

Control reactions of HDL treated with H₂O₂ alone showed no decrease inABCA1-dependent cholesterol efflux, demonstrating the critical nature ofthe MPO-catalyzed peroxidase reaction. The same tyrosine residues werealso modified and a similar pattern of decreased ABCA1-dependentcholesterol efflux were seen control experiments using lipid-free apoA-Itreated with the complete MPO-mediated modification systems (data notshown). These results are consistent with protein modification, asopposed to lipid modification, being responsible for the loss of effluxactivity.

The effect of MPO-mediated modification on the lipid bindingcharacteristics of apoA-I was also tested. These experiments measure thelipid binding activity of apoA-I by monitoring the ability of the apoA-Ito inhibit the aggregation of LDL that is treated with phospholipase C(PLC). The apoA-I inhibition is due to its ability to coat the modifiedhydrophobic LDL through a lipid binding process that is an initial stepin apoA-I-mediated cholesterol efflux. As shown in FIG. 6, LDL treatedwith the PLC produces a time-dependent aggregation that is significantlyreduced by the lipid binding activity of unmodified apoA-I or apoA-Ithat was pretreated with hydrogen peroxide alone (FIG. 9A). Pretreatmentof the apoA-I with the MPO-mediated nitration and chlorination systems(MPO/H₂O₂/NO₂ ⁻ and MPO/H₂O₂/Cl⁻, respectively) significantly inhibitedthis reduction with the nitration reaction giving a 10% inhibition andthe chlorination reaction giving a 35% inhibition. The identicalmodified or control apoA-I preparations were tested for ABCA1-dependentlipid efflux acceptor activity and the decreases in apoA-I's lipidbinding activity correlated directly with observed losses ofABCA1-dependent efflux acceptor activity (FIG. 9B).

Example 7 Specific apoA-I Modification Sites are Found In Vivo

The LC-tandem MS studies of apoA-I modified in vitro by MPO, describedabove, produced a roster of modification sites for evaluation in vivo.FIG. 7 shows a series of SRM chromatograms from the LC-tandem MSanalysis of apoA-I that was isolated from human atheroma tissues. Theelution of peptides containing nitration at the two primary nitrationsites, Y192 and Y166, are seen in the chromatograms shown FIGS. 7A and7C, respectively. The CID spectra (FIGS. 10B and 10D, respectively)recorded at these retention times provide unambiguous proof of thecorrect identify of these nitrated peptides. The amounts of the nitratedpeptides, relative to the respective un-modified peptides, can beestimated as 9% for the Y192-containing peptide and 0.2% for theY166-containing peptide by integrating the area of each chromatographicpeak. These values must be considered estimates because the relativeLC-MS responses of the nitrated versus un-nitrated peptides have notbeen determined. Similar experiments targeting the secondary nitrationsites identified through the in vitro experiments could not detect theY29- and Y236-containing peptides in the nitrated forms (data notshown). This inability to detect these nitrated peptides is consistentwith the relatively poor efficiency of the MPO-mediated nitration ofthese sites.

We also attempted to verify the presence of site-specific chlorinatedpeptides. A peptide containing chlorination at the Y192 position couldnot be detected (data not shown) despite the identification of this siteas the preferred site of chlorination in in vitro models. The peptidecontaining chlorination at the Y166 position could be detected (bottompanel FIG. 7B), although the CID spectrum (bottom panel FIG. 7D) showeda superimposed spectrum of peptide ions containing chlorination of Y166and oxidation of Y166 to give trihydroxyphenylalanine. This oxidizedpeptide has a molecular weight that is 2 Da lower than the correspondingchlorinated species. As a doubly charged ion, however, the chlorinatedand oxidized peptides differ by 1 Da in the m/z scale of the massspectrometer. This m/z difference cannot be distinguished in the 2 Daacceptance window of the first stage of mass analysis in the ion trapdetector. The resulting singly charged fragment ions differ by 2 Da andcan be distinguished in the CID spectrum.

1. A method for characterizing a subject's risk of having cardiovasculardisease, comprising: determining levels of one or more oxidizedapolipoprotein A-I (apoA-I)-related biomolecules in a biological samplefrom the subject, wherein the one or more oxidized apoA-I-relatedbiomolecules are selected from oxidized HDL, oxidized apoA-I, and anoxidized apoA-I peptide fragment, and wherein elevated levels of the oneor more apoA-I-related oxidized biomolecules in the biological sample ascompared to a control value or an internal standard indicates that thesubject is at risk of having cardiovascular disease.
 2. The method ofclaim 1, wherein at least one of the one or more oxidized apoA-I-relatedbiomolecules is oxidized apoA-I or an oxidized apoA-I peptide fragment.3. The method of claim 2, wherein the method employs a procedure orreagent for detecting oxidized apoA-I or an oxidized apoA-I peptidefragment that comprises one or more of the following amino acidresidues: chlorotyrosine, nitrotyrosine, dityrosine,trihydroxy-phenylalanine, dihydroxyphenylalanine, methionine sulphoxide,and tyrosine peroxide.
 4. The method of claim 1, wherein the methodemploys a procedure for detecting oxidized apoA-I or an oxidized apoA-Ipeptide that comprises an oxidized amino acid residue at any one ofpositions 18, 29, 166, 192, 236, or any combination of said positions,in SEQ ID NO:
 1. 5. The method of claim 1, wherein levels of the one ormore oxidized apoA-1 related biomolecules are compared to a controlvalue or a range of control values based upon levels of the one or moreoxidized apoA-I-related biomolecules in comparable biological samplesfrom a control population of human subjects.
 6. The method of claim 1,wherein said biological sample is blood, serum, or plasma.
 7. A methodfor characterizing a subject's risk profile for cardiovascular disease,comprising: determining a first risk value by comparing levels of one ormore oxidized apoA-I-related biomolecules in a bodily sample from thesubject to a control value; and determining one or more additionalcardiovascular risk values in the subject, wherein said one or moreadditional risk values are obtained by a) determining the subject'sblood pressure; b) determining levels of low density lipoprotein, orcholesterol, or both in a biological sample from the subject; c)assessing the subject's response to a stress test; d) determining levelsof myeloperoxidase, C-reactive protein, or both in a biological samplefrom the subject; and e) determining the subject's atheroscleroticplaque burden, and combining said first risk value with said one or moreadditional risk values to provide a final risk value.
 8. A method forcharacterizing a subject's risk of developing cardiovascular disease,comprising: determining levels of one or more oxidized apoA-I relatedbiomolecules in a biological sample from the subject, wherein the one ormore apoA-I-related biomolecules are selected from oxidized HDL,oxidized apoA-I, and an oxidized apoA-I peptide fragment, and whereinelevated levels of the one or more apoA-I-related oxidized biomoleculesin the biological sample as compared to a control value or an internalstandard indicates that the subject is at risk of developingcardiovascular disease.
 9. The method of claim 8, wherein at least oneof said one or more oxidized apoA-I-related biomolecules is oxidizedapoA-I or an oxidized apoA-I peptide fragment.
 10. The method of claim8, wherein the method employs a procedure or reagent for detectingoxidized apoA-I or an oxidized apoA-I peptide fragment that comprisesone or more of the following amino acid residues: chlorotyrosine,nitrotyrosine, dityrosine, trihydroxy-phenylalanine,dihydroxyphenylalanine, methionine sulphoxide, and tyrosine peroxide.11. The method of claim 8, wherein the method employs a procedure fordetecting oxidized apoA-I or an oxidized apoA-I peptide that comprisesan oxidized amino acid residue at any one of positions 18, 29, 166, 192,236, or any combination of said positions, in SEQ ID NO:
 1. 12. Themethod of claim 8, wherein levels of the one or more oxidized apoA-Irelated biomolecules are compared to a control value or a range ofcontrol values based upon levels of the one or more oxidizedapoA-I-related biomolecules in comparable biological samples from acontrol population of human subjects.
 13. The method of claim 8, whereinsaid biological sample is blood, serum, or plasma.
 14. The method ofclaim 8, wherein the subject is an apparently healthy human subject. 15.The method of claim 14, wherein the subject is a non-smoker.
 16. Themethod of claim 8, wherein the subject is not otherwise known to be atan elevated risk of having cardiovascular disease.
 17. A method forcharacterizing the near term risk of experiencing a major adversecardiac event in a subject who is presenting with chest pain,comprising: determining levels of one or more oxidized apoA-I-relatedbiomolecules in a biological sample from the subject, wherein said oneor more apoA-I-related biomolecules is selected from oxidized HDL,oxidized apoA-I, and an oxidized apoA-I peptide fragment, and whereinelevated levels of the one or more apoA-I-related oxidized biomoleculesin the biological sample as compared to a control value or an internalstandard indicates that the subject is at risk of experiencing a majorcardiac event.
 18. The method of claim 17, wherein at least one of saidone or more oxidized apoA-I-related biomolecules is oxidized apoA-I oran oxidized apoA-I peptide fragment.
 19. The method of claim 17, whereinthe method employs a procedure or reagent for detecting oxidized apoA-Ior an oxidized apoA-I peptide fragment that comprises one or more of thefollowing amino acid residues: chlorotyrosine, nitrotyrosine,dityrosine, trihydroxy-phenylalanine, dihydroxyphenylalanine, methioninesulphoxide, and tyrosine peroxide.
 20. The method of claim 17, whereinthe method employs a procedure for detecting oxidized apoA-I or anoxidized apoA-I peptide that comprises an oxidized amino acid residue atany one of positions 18, 29, 166, 192, 236, or any combination of saidpositions, in SEQ ID NO:
 1. 21. The method of claim 17, wherein levelsof the one or more oxidized apoA-I related biomolecules are compared toa control value or a range of control values based upon levels of theone or more oxidized apoA-I-related biomolecules in comparablebiological samples from a control population of human subjects.
 22. Themethod of claim 17, wherein said biological sample is blood, serum, orplasma.
 23. A method for evaluating therapy in a subject suspected ofhaving or diagnosed as having cardiovascular disease, comprising:determining levels of one or more oxidized apoA-I-related biomoleculesin a biological sample taken from the subject prior to therapy anddetermining levels of the one or more of the oxidized apoA-I relatedbiomolecules in a corresponding biological sample taken from the subjectduring or following therapy, wherein a decrease in levels of the one ormore oxidized apoA-I-related biomolecules in the sample taken after orduring therapy as compared to levels of the one or more oxidizedapo-I-related biomolecules in the sample taken before therapy isindicative of a positive effect of the therapy on cardiovascular diseasein the treated subject.
 24. The method of claim 13, wherein saidbiological sample is blood, serum, or plasma.
 25. A method of assessinga subject's risk of experiencing a complication of cardiovasculardisease, comprising comparing levels of one or more oxidized forms of abiomolecule in a biological sample from the subject with levels of saidone or more oxidized forms of said biomolecule in a biological samplefrom a control population of subjects; wherein said biomolecule isoxidized HDL, oxidized apoA-I, one or more oxidized apoA-I peptidefragments, or combinations thereof, and characterizing the subject'srisk of experiencing a complication of cardiovascular disease based uponlevels of the one or more oxidized biomolecule in the subject's bodilysample in comparison to a control value derived from a controlpopulation of subjects.
 26. The method of claim 25, wherein thedifference between levels of said one or more oxidized forms of abiomolecule in a biological sample from the subject as compared tolevels of said one or more oxidized forms of said biomolecule biologicalsamples from the control population of subjects is indicative of theextent of the subject's risk of experiencing a complication ofcardiovascular disease.
 27. The method of claim 25, wherein the methodemploys a procedure or reagent for detecting oxidized apoA-I or anoxidized apoA-I peptide fragment that comprises one or more of thefollowing amino acid residues: chlorotyrosine, nitrotyrosine,dityrosine, trihydroxy-phenylalanine, dihydroxyphenylalanine, methioninesulphoxide residue, and tyrosine peroxide.
 28. The method of claim 25,wherein the sample is blood or a fraction thereof.
 29. A method ofdetermining the near term risk of requiring medical intervention in asubject presenting with chest pain, comprising: assessing the level of arisk predictor in a bodily sample from the subject, wherein said riskpredictor is oxidized HDL, oxidized apoA-I, an oxidized apoA-I peptidefragment, or any combination thereof, wherein the difference between thelevel of the risk predictor in the subject's bodily sample and the levelof the risk predictor in a comparable bodily sample from the controlpopulation establishes the extent of the risk of requiring medicalintervention near term in the subject.
 30. A method of characterizingthe risk of experiencing a subsequent acute cardiovascular event in asubject who has experienced one or more acute adverse cardiovascularevents, comprising: determining levels of one or more of the oxidizedapoA-I-related biomolecules in a biological sample taken from thesubject at an initial time and in a corresponding biological sampletaken from the subject at a subsequent time; wherein an increase inlevels of the one or more oxidized apoA-I-related biomolecules in abiological sample taken at the subsequent time as compared to theinitial time indicates that a subject's risk of experiencing asubsequent adverse cardiovascular event has increased.
 31. A method formonitoring over time the status of cardiovascular disease (CVD) in asubject with CVD, comprising: determining the levels of one or more ofthe oxidized apoA-I-related biomolecules in a biological sample takenfrom the subject at an initial time and in a corresponding biologicalsample taken from the subject at a subsequent time, wherein am increasein levels of the one or more oxidized apoA-I-related biomolecules in abiological sample taken at the subsequent time as compared to theinitial time indicates that a subject's CVD has worsened.
 32. Anantibody immunospecific for oxidized apoA-I, or an oxidized apoA-Ipeptide fragment, wherein said apoA-I peptide fragment is at least threeamino acids in length and comprises one or more of the following aminoacid residues: chlorotyrosine, nitrotyrosine, dityrosine,trihydroxyphenylalanine, dihydroxyphenylalanine, methionine sulphoxideresidue, and tyrosine peroxide.
 33. The antibody of claim 32, whereinthe oxidized apoA-I protein or peptide fragment comprises at least onechlorotyrosine.
 34. The antibody of claim 32, wherein the oxidizedapoA-I protein or peptide fragment comprises at least one nitrotyrosine.35. The antibody of claim 32, wherein the oxidized apoA-I protein orpeptide fragment comprises at least one dihydroxyphenylalanine.
 36. Theantibody of claim 32, wherein the oxidized apoA-I protein or peptidefragment comprises at least one trihydroxyphenylalanine.
 37. Theantibody of claim 32, wherein the oxidized apoA-I protein or peptidefragment comprises at least one dityrosine.
 38. The antibody of claim32, wherein the oxidized apoA-I protein or peptide fragment comprises atleast one methoinine sulfoxide.
 39. The antibody of claim 32, whereinthe oxidized apoA-I protein or peptide fragment comprises at least onetyrosine peroxide.
 40. A kit comprising: one or more reagents fordetecting oxidized apoA-I or an oxidized fragment of apoA-I, and one ormore of the following printed materials: instructions for using saidreagent in a method of assessing a test subject's risk of cardiovasculardisease, information for assessing a test subjects risk cardiovasculardisease, and recommendations for treating a subject who is determined tobe at risk of cardiovascular disease.
 41. The kit of claim 40, whereinat least one of the one or more reagents is an antibody that isimmunospecific for oxidized apoA-I or an oxidized peptide fragment ofapoA-I, or both.
 42. The kit of claim 40, wherein at least one of theone or more reagents is oxidized HDL, oxidized apoA-I, an oxidizedapoA-I peptide fragment.
 43. A method of treating a person suspected ofhaving or diagnosed as having cardiovascular disease, comprising:determining levels of one or more oxidized apoA-I-related biomoleculesin a biological sample from the subject, wherein said one or moreapoA-I-related biomolecules is selected from oxidized HDL, oxidizedapoA-I, and an oxidized apoA-I peptide fragment, and where the levels ofthe one or more oxidized apoA-I-related biomolecules are elevated ascompared to a control sample, providing interventional treatment toreduce the person's risk of a near term adverse cardiac event.
 44. Themethod according to claim 43, wherein the interventional treatment ischosen from; recommending medication for cardiovascular disease for thesubject, recommending coronary angiography for the subject, recommendingcoronary by-pass surgery for the subject.
 45. A method of treating aperson suspected of having or diagnosed as having cardiovasculardisease, comprising: determining levels of one or more oxidizedapoA-I-related biomolecules in a biological sample from the subject,wherein said one or more apoA-I-related biomolecules is selected fromoxidized HDL, oxidized apoA-I, and an oxidized apoA-I peptide fragment,and where the levels of the one or more oxidized apoA-I-relatedbiomolecules are elevated as compared to a control sample, providingadvice to the person to improve the person's long term cardiovascularhealth.
 46. A method for treating a subject to reduce the risk of acardiovascular disorder, comprising: determining levels of one or moreoxidized apoA-I-related biomolecules in a biological sample from thesubject, wherein said one or more apoA-I-related biomolecules isselected from oxidized HDL, oxidized apoA-I, and an oxidized apoA-Ipeptide fragment, and where the levels of the one or more oxidizedapoA-I-related biomolecules are elevated as compared to a control samplean agent chosen from an anti-inflammatory agent, an antithromboticagent, an anti-platelet agent, a fibrinolytic agent, a lipid reducingagent, a direct thrombin inhibitor, a glycoprotein IIb/IIIa receptorinhibitor, an agent that binds to cellular adhesion molecules andinhibits the ability of white blood cells to attach to such molecules, acalcium channel blocker, a beta-adrenergic receptor blocker, acyclooxygenase-2 inhibitor, or an angiotensin system inhibitor, isadministered to the subject.
 47. The method of claim 46, wherein thesubject is otherwise free of symptoms calling for treatment with theagent.
 48. The method of claim 46, wherein the subject is apparentlyhealthy and does not otherwise have an elevated risk of an adversecardiovascular event.
 49. The method of claim 46, wherein the subject isnon-hyperlipidemic.
 50. The method of claim 46, wherein the agent is anon-aspirin, anti-inflammatory agent.